CHARACTERIZATION AND REGULATION OF HUMAN STEAROYL-COA

DESATURASE 5

by

GRETCHUN JUNGYUN KIM

A Thesis submitted to the

Graduate School - New Brunswick

Rutgers, The State University of New Jersey

in partial fulfillment of the requirements

for the degree of

Master of Science

Graduate Program in Food Science

written under the direction of

Dr. R. Ariel Igal

and approved by

_____________________________

_____________________________

_____________________________

New Brunswick, New Jersey

October 2011

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ABSTRACT OF THE THESIS

Characterization and regulation of human Stearoyl-CoA Desaturase 5

By GRETCHUN JUNGYUN KIM

Thesis Director: Dr. R. Ariel Igal, M.D., Ph.D

Saturated (SFA) and monounsaturated fatty acids (MUFA) are among the

most abundant fatty acids in mammalian organisms. These fatty acids are

fundamental components of structural, energetic, and signaling lipids; hence their

levels have to be tightly regulated by the cell. The abundance of SFA and MUFA

is determined by the rate of activity of Stearoyl-CoA Desaturase (SCD), an

enzyme responsible for biosynthesis of MUFA by inserting a double bond of a

saturated acyl-CoA. To date, four isoforms of SCD have been described in

murine, and two isoforms in humans. Unlike ubiquitously expressed SCD1,

human SCD5 is predominantly expressed in brains and pancreas. SCD1 is a

critical factor of the control of cell lipogenesis, as well as cell proliferation and

differentiation. Importantly, dysfunctional SCD1 is linked to the onset of several

widespread diseases, including obesity, diabetes, and cancer. Despite

considerable amount of research devoted to unravel the mechanisms of SCD1

regulation, the role and regulation of SCD5 is virtually unknown.

In the present study, we determined the abundance of SCD5 protein in

human cell lines including normal, transformed, and lung cancer cell lines.

Further, we examined the role of insulin and epidermal growth factor in the

regulation of SCD5 levels in normal and neoplastic human cells. Finally, since

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the high levels of SCD5 found in the brain suggest a possible function of this

enzyme in neuronal differentiation, we examined the effect of retinoic acid on the

content of SCD5 in human neuroblastoma cells. Here, we report that SCD5 is

abundantly expressed in different human cell types and cell lines, and that SCD5

levels are increased by insulin, likely through the activation of the PI3K/Akt

pathway, a signaling cascade that controls lipid synthesis, mitogenesis and

tumorigenesis. In addition, we found a slight increased SCD5 during the process

of neuronal differentiation. Thus, our findings demonstrate that the levels of

SCD5 are modulated by critical factors that control cell growth, survival and

differentiation in human cells. Taken together, these data suggests SCD as

potential target for pharmacological and nutritional interventions in a number of

human diseases and conditions.

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ACKNOWLEDGMENTS

This research and educational journey at Rutgers University would not have

been possible without kind support and guidance of many people. I would like to

sincerely extend my gratitude to my advisor, R. Ariel Igal, for his openness and patience

guidance. I would also like to sincerely thank my graduate advisory committee, Drs.

George Carman, Loredana Quadro and Nanjoo Suh for their valuable time, thoughtful

advice, and helpful discussions.

I am also greatly indebted to my friends and members and nonofficial members

of Igal’s lab for their thoughtful scientific discussions, ideas, humor, and support during

our tea breaks. I am forever indebted to Dr. Mark Failla at the Ohio State University who

has first introduced me to discover passion and excitement through research. I am also

indebted to Seung-Shick Shin, Sagar Thakkar, and Tianyao Huo for their absolute

support and guidance. Their unconditional support and friendship is greatly appreciated

and embedded deeply in my heart.

I would like to acknowledge the following organizations as source of material

support: Johnson & Johnson Centocor R&D, Brent Rupnow (Bristol-Myers Squibb), and

School of Environmental and Biological Sciences and New Jersey Agricultural Station.

Additionally, this journey to higher education would not have been possible without

financial supports from U.S. Department of Education and Federal Student Aid.

Lastly and most importantly, I would like to thank my parents, Sue Kim, Chanel

Sanchez, Eric Hamrick, and Angelus for their endless love and encouragement

throughout the graduate program.

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TABLE OF CONTENT

ABSTRACT……………………………………………………….…….………………..ii

ACKNOWLEDGEMENTS……………………………………….…….……………….iv

TABLE OF CONTENT …………………………………………….…….……………..v

LIST OF TABLES…………………………………………………………..…………..vi

LIST OF FIGURES………………………………………………….…………………vii

LIST OF ABBREVIATIONS………………………………………….…….…………viii

INTRODUCTION…………………………………………………….……….…………1

RESEARCH AIMS……………………………………………………………..………17

MATERIALS AND METHODS………………………………………………….……20

RESULTS……………………………………………………………………..….…….26

DISCUSSIONS……………………………………………….……………………..…35

CONCLUSIONS…………………………………………………………….....…..…..39

FUTURE WORKS………………………………………………………….……….....40

REFERENCES…………………………………………………………………………41

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LIST OF TABLES

Table 1. Regulation of SCD1 by dietary factors……………………………………12

Table 2. Regulation of SCD1 by hormones…………………………………..…….13

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LIST OF FIGURES

Figure 1. Regulation of MUFA/SFA balance in mammalian cell by SCD1...........4 Figure 2. Biosynthesis of MUFA by SCD……………………………………….…...6 Figure 3. Topology protein model of mouse SCD1.......................................…...6 Figure 4. Verification of anti-SCD5 antibody efficacy…………..………………...27 Figure 5. SCD5 protein is ubiquitously expressed in various human cell lines...29 Figure 6. Effect of growth factors on the expression of SCD1 and SCD5 across cell types and cell lines……………………………………………………32 Figure 7. Effect of RA induction in neuronal differentiation on SCD5………..…34

viii

LIST OF ABBREVIATIONS

ACP Acyl-carrier protein

Akt Serine/threonin protein kinase Akt

ACOD4 Acyl CoA desaturase 4

CE Cholesteryl ester

CLA Conjugated lioleic acid

DMSO Dimethyl sulfoxide

ER Endoplasmic reticulum

ERK Extracellular signal-regulated kinases

EGF Epidermal growth factor

FBS Fetal bovine serum

GAP-43 Growth associated protein 43

INS Insulin

MAP-2 Microtubule-associated protein 2

MAPK Mitogen activated protein kinase

MUFA Monounsaturated fatty acid

NADPH Nicotinamide adenine dinucleotide phosphate reduced hydrogen

PI3K Phosphoinositide 3-kinase

PL Phospholipids

PUFA Polyunsaturated fatty acid

RA Retinoic acid

SCD Stearyl CoA desaturase

SFA Saturated fatty acid

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SREBP-1 Stearol regulatory element-binding protein-1

TAG Triacylglycerols

UFA Unsaturated fatty acid

1

INTRODUCTION

Out of four macromolecule building blocks of life (nucleic acids,

carbohydrates, proteins, and lipids), lipids contribute many key roles in diverse

biological functions. Main fatty acid-containing lipids, such as phospholipids,

diacylglycerols, and triacylglycerols display a variety of biological roles including

structural, signaling and energetic (Figure 1). Among the fatty acids esterified to

backbones of these lipids, saturated fatty acids (SFA) and monounsaturated fatty

acids (MUFA) are the most abundant acyl groups in mammalian cells (Igal 2010).

Also, in growing yeast, MUFA alone contribute up to 80% of the fatty acid content

in membrane lipids (Martin et al., 2007). Similarly, in humans SFA and MUFA

make up to 78.4 % of all fatty acid groups in triacylglycerols (Hodson et al., 2008).

It is interesting to note that although mammalian cells can incorporate both SFA

and MUFA, proliferating mammalian cells, particularly cancer cells,

endogenously produce these fatty acids for the synthesis of complex lipids

(Sabine et al., 1967, Ookhtens et al., 1984). MUFA are also functionally critical

fatty acids non-mammalian eukaryotic cells. Yeast cells (Saccharomyces

cerevisiae and Schizosaccharomyces pombe), for instance, can biosynthesize all

the needed lipids for supporting cell growth under aerobic condition (Martin et al.,

2007) and endogenously produced MUFA are fundamental for these processes

(Weete 1974, McDonough et al., 2004).

Because the balance between SFA and MUFA is critical for many cellular

functions, cells must tightly control their levels. A major point of this regulation is

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the control of the biosynthesis of MUFA by stearoyl-CoA desaturase (SCD). In

eukaryotic cells, SFA are synthesized by two committed-steps involving the

formation of malonyl-CoA by acetyl-CoA carboxylase in the cytosol,followed by

the biosynthesis of the SFA palmitate (16:0) by the fatty acid synthase enzyme

complex, using maloyl-CoA and acetyl-CoA. Finally, the biosynthesis of MUFA is

catalyzed by SCD (cis-Δ9 desaturase, also known as Ole1p in yeast). The SCD

is a family of endoplasmic reticulum-resident enzymes that introduce a first

double bond in the cis-Δ9 position of a saturated fatty acyl-CoAs (Enoch et al.,

1976). Although Enoch et al.,1976 reported that the purified rat SCD catalyze

the desaturation of 12 to 18-carbon fatty acids, the preferred saturated fatty-acyl

CoA substrates for SCD are palmitoyl-CoA (16:0) and stearoyl-CoA (18:0)

(Enoch et al., 1976:1978, Mahfouz et al., 1980, Strittmatter et al.,1978, Ntambi et

al.,1995, Ntambi et al., 1999). It is also important to note that SCD are able to

catalyze desaturation of both exogenous and endogenously synthesized SFA

(Igal 2010). Nevertheless, in both normal and cancer cells in culture, the levels of

MUFA in cells are mainly determined by the rate of SCD activity (Igal 2010).

Over the years, studies have demonstrated that different SCD isoforms in

mice are responsible for biosynthesis of MUFA (Ntambi et al., 2004). So far,

there are up to four isoforms of SCD (SCD1, SCD2, SCD3, and SCD4)

characterized and described in mice (Miyazaki et al., 2001, Kaestner et al., 1989,

Ntambi et al., 1988, Miyazaki et al., 2003a:b), whereas only two isoforms of SCD,

SCD1 and SCD5 (also known as ACOD4), were identified in humans (Zhang et

al., 1999, Beiraghi et al., 2003, Wang et al., 2005). Mouse SCD isoforms share

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high percentage of homology in their amino acid sequence and all of those

isoforms are produced by genes that are all located in close proximity on a single

chromosome (Zheng et al., 2001, Tabor et al., 1998). In fact, unlike mice SCD

isoforms in which all genes of SCD isoforms are located less than 200 kb apart

from one another on chromosome 19, human SCD1 and SCD5 genes are

located on two separate chromosomes, chromosome 10 and chromosome 4,

respectively (Wang et al., 2005). Although some studies suggest that tissue-

specific expression of SCD in mice may be part of a local regulatory pathway

(Miyazaki et al., 2003a:b), the potential advantage of tissue-specific isoforms of

SCD remains unknown.

In human tissues, SCD1 is present in most tissues, but it is particularly in

high levels in liver, adipose tissue, lung and brain tissue (Wang et al., 2005).

SCD5, in turn, is highly expressed in adult brain and in embryo tissues,

especially in brain and pancreas, suggesting a potential specific role for SCD5 in

those tissues. MUFA, specifically oleic acid (18:1), are abundantly synthesized in

the brain cells by SCD activity and these fatty acids are known to act as a

neurotrophic factors (Tabernero et al., 2001). Since Wang et al., 2005 have

demonstrated that SCD5 is the predominant isoform in fetal brain tissues,

whereas in adults brain samples have somewhat of an equal representation of

both SCD1 and SCD5 isoforms, these two isoforms may contribute with MUFA

for brain functions. Currently, there are only a few studies have been conducted

using human tissue samples to characterize and identify SCD5.The regulation of

human SCD5 is virtually unknown. Therefore, the goal of this project was to

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initiate the characterization of SCD levels in human normal and cancer cells, and

investigate the regulation of human SCD enzymes, especially SCD5 protein, by

growth factors that are critical for the regulation of lipid synthesis, cell

proliferation and survival.

Figure 1. Regulation of MUFA/SFA balance in mammalian cell lipids by SCD1. CE, cholesterol esters; PL, phospholipids: TAG, triacylglycerol. (Igal 2010)

Stearoyl- CoA desaturases: function and regulation

Mammalian SCD are exclusively located in the endoplasmic reticulum

compartment. Structurally, SCD protein contains four transmembrane domains

that are embedded in the endoplasmic reticulum, whereas the catalytically

important three conserved histidine motif box regions are oriented toward the

cytosol side (Stukey et al., 1990, Shaklin et al., 1994, Man et al., 2006). The

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desaturating reaction is illustrated in Figure 2. Briefly, two electrons and two

protons donated by NAD(P)H+H+ are accepted by cytochrome b5 reductase and

passed to heme iron of cytochrome b5, which then donates the electrons to SCD,

and lastly to oxygen. In the process, SCD introduces a double bond into a

saturated hydrogen carbon chain by removing two electrons and two protons

from adjacent methylene groups and transferring them to oxygen and forming

water (Fox et al., 2004). This SCD catalytic mechanism is supported by studies

done with plant chloroplast Δ9 desaturases, in which soluble saturated fatty acyl-

carrier protein (ACP) is used as substrate (Fox et al., 2004, Shaklin et al.,1998),

and by investigations of yeast integral membrane desaturases (Behrouzian et al.,

2003). The desaturation is an energetically consuming process costing

approximately 397.82kJ/mol (Fox et al., 2004).

The yeast cell gene OLE1 (Gene ID: 852825) encodes its yeast cell

desaturase protein (Ole1p), which is located in the endoplastimic reticulum with

strikingly similar structure features of mammalian SCD. These features include

location and number of histidine motif boxes, four segments of aligned regions of

genes sharing greater than 70% identity, and presence of two long hydrophobic

sequences (transmembrane loops) in the positions of amino acid sequence of

cloned rat and yeast gene samples (Stukey et al., 1990 Martin et al., 2007).

However, unlike mammalian SCD1, yeast Ole1p desaturase contains

cytochrome b5 linked to C-terminus of Ole1p as an integral part of its protein

(Mitchell et al., 1995). Moreover, Martin et al., 2002 reported that similar to

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mammalian Δ9 desaturases, Ole1p desaturase can used a wide range of fatty

acyl CoA substrates.

Figure 1. Biosynthesis of MUFA by SCD (Ntambi et al., 2003)

Figure 2. Topology protein model of mouse SCD1 (Ntambi et al., 2003)

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Identification of Stearoyl-CoA desaturase-5, a novel SCD isoform

SCD5 (also known as ACOD4) is the most recently discovered SCD

isoform. Originally, ACOD4 was first identified and published as a novel acyl-CoA

desaturase enzyme located in chromosome 4q21 where pericentric inversion

breakpoint that disrupts SCD5 gene was reported to be present in both father

and a son in a family with history of cleft lip (Beiraghi et al., 2003). In this seminal

work, Beiraghi et al., 2003 also reported that the mRNA level of SCD5 is highly

expressed in fetal brain and lower level in fetal kidney across human tissues.

Two years after Beiraghi et al., 2003 publication, Wang et al., 2005 reported the

presence of SCD5 mRNA in other human fetal tissues such as kidney, lung,

spleen, and thymus. Using SCD5-overexpressing cells, these authors

unambiguously demonstrated that a) SCD5 exhibits ∆9-desaturating activity, b) it

is able to use both palmitic and stearic acid as substrates, and c) like SCD1, is

exclusively located in the endoplasmic reticulum of the cell (Wang et al., 2005).

Besides these initial studies on the identification of SCD5 as novel SCD in

humans, recent reports indicate that this isoform is also present in non-primate

mammals such as dogs, cattle, and sheep. In the case of ruminants, early

studies by Kinsella et al., 1972 reported abundantly expressed SCD1 in

mammary glands of lactating cows, suggesting that this enzyme may play a role

in mediating milk production (Martin et al., 1999, St John et al., 1991, Ward et al.,

1998). In 2007, a SCD5 gene was identified in bovine tissues (Lengi et al., 2007).

These authors found that mRNA expression of SCD5 and SCD1 were detectable

in adipose tissue, brain, heart, liver, lung, muscle tissues and that expression of

8

SCD5 in bovine tissues was highest in the brain, whereas highest expression of

SCD1 was observed in adipose tissue (Lengi et al., 2007). Importantly, Gervais

et al., 2009 showed the presence of high SCD5 expression, in conjunction with

SCD1, in mammary gland of lactating cows suggesting that the novel desaturase

may also participate in the process of milk production. Furthermore, SCD5 in

mammary gland of cows appears to be regulated differently than SCD1 by

nutrients during lactation (Gervais et al., 2009). In this connection, Jacobs et al.,

2011 observed that when lactating cows were supplemented with plant oils such

as rapeseed oil (rich in 18:1), soybean oil (rich in 18:2), linseed oil (rich in 18:3),

and mixture of these three oils in 1:1:1 ratio, the soybean oil- supplemented

group had a significant decrease in SCD1 mRNA level in their mammary gland

tissue. However, they found that SCD5 mRNA levels in these tissues were not

statistically significant among all four plant oil supplementations in lactating cows

suggesting that SCD5 may be less sensitive to nutritional regulation than SCD1

(Jacobs et al., 2011). Further reinforcing this concept, it was observed that while

dietary PUFA (including CLA) downregulated SCD1 expression in mammary

glands of cows, a similar treatment did not affect SCD5 expression levels

(Jacobs, et al., 2011).

Revealing the regulatory mechanisms of SCD5 may provide answers to

the role of the desaturases in a number of biological functions but also novel

technological tools for the modification of food quality. Due to increased research

studies to support the health benefits of unsaturated fatty acids (UFA), efforts to

increase these fatty acids, particularly MUFA, in milk fat have attracted

9

researchers into studying one of the major players of MUFA biosynthesis in dairy

cows, SCD. Bovine milk fats, mainly as triacylglycerols, are composed of over

400 molecular species (Lindamark-Månsson 2008). Reportedly, Swedish dairy

milk fat contains 69.4% and 30.6% of total milk fat as SFA and UFA, respectively,

in which 25% of those UFA are MUFA, with oleic acid representing 23.8% of

percent by weight of the total fatty acids (Lindamark-Månsson 2008).

Role of MUFA in neuronal differentiation

The synthesis of MUFA catalyzed by SCD1 has been shown to be critical

for the processes of cell proliferation and adipose cell differentiation (Igal 2010,

Kasturi et al., 1982, Ntambi et al., 1988). The presence of both SCD1 and the

newly discovered SCD5 in developing brains point to a potential role of these

desaturases in neuronal growth, differentiation and in brain formation. A typical

marker of neuronal differentiation in neuroblast cells in culture is observed when

long projections start emerging from the cell body. These cell projections can be

either dendrites or axons. In cell models of neuronal differentiation, such as

mouse Neuro2A and human SH-SY5Y Neuroblastoma cells, upon stimulation

with retinoic acid (RA) will result in growth and network of dendrites emerging

from the cell body.

The differentiation process is characterized by modifications in lipid

biosynthesis, especially by increased levels of membrane phospholipid synthesis

(Marcucci et al., 2010), likely to provide lipid structures for the formation of

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neurites. The biosynthesis of MUFA and, most importantly, the role of SCD5 in

the mechanisms of differentiation have not been explored to date.

The formation of new lipids for new cellular membranes and for myelin

sheath in differentiating neurons has been shown to be a mandatory event

(Garbay et al., 2000). The myelin sheath is one of the most tightly cored multiple

membrane layer component structure of a functional neuron cell around the axon

of neuron cells. Its primary function is to provide insulation for the nerve fiber and

facilitate the nerve impulse conduction. In both mammals and non-mammal

species, the lipid accounts for 72-78% whereas proteins represent between 20-

30% dried myelin mass in peripheral nervous system (Garbay et al., 2000). Out

of various fatty acid composition, oleic acid (18:1) represents the predominant

fatty acid species in myelin sheath such that it comprises between 30 -40% of all

fatty acids in sciatic nerves, the single largest and longest nerve fiber in human

body (Garbay et al., 2000). Tabernero et al., 2001 demonstrated that the

presence of oleic acid induces axonal growth, neuronal clustering, and the up-

regulation of molecular markers of axonal and dendritic growth, such as GAP-43

and MAP-2. A positive correlation between SCD1 protein expression and a

molecular marker (GAP-43) for axonal growth was made to confirm the

involvement of SCD in neuronal differentiation (Polo-Hernandez et al., 2010).

Furthermore, these oleic-induced changes are Protein Kinase C-dependent

(Tabernero et al., 2001: 2002). Because oleic acid is one of major products of

SCD activity, the neurotropic function of oleic acid in neuronal differentiation may

be in the end determined by the rate of SCD expression and activity. Indeed,

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Garbay et al., 1998:2000 reported a high activity level of SCD in normal mouse

sciatic nerves during the first three weeks after birth, whereas trembler mice (an

animal model for peripheral neuropathy) showed very low SCD activity, further

strengthening a link between high SCD activity, neuronal development and

myelin synthesis in the peripheral nervous system. Out of four SCD isoforms in

mice, SCD2 is the major desaturase in which its enzyme activity contributes to

high oleic acid production in the brain (Garbay et al., 1998: 2000). Interestingly,

the regulation of SCD2 expression of mRNA has shown to share parallel

regulation as myelin-specific genes in peripheral nerve system involved in

postnatal nerve development and nerve degeneration (Garbay et al., 2000).

Since SCD2, albeit the lack of sequence homology, exhibit a similar tissues

distribution than human SCD5, it is tempting to speculate that both desaturases

may play specific roles in neuronal development and differentiation in their

respective species.

Regulation of Stearoyl-CoA desaturases by nutritional, hormonal, and

growth factors

SCD1 expression is remarkably sensitive to myriad nutrients, including

carbohydrates, fatty acids, and cholesterol, as well as a great number of

hormones and growth factors (Ntambi et al., 2004). For instance, as described in

Table 1, expression of hepatic SCD1 is induced by glucose, fructose, vitamin A,

cholesterol, and vitamin D, whereas it is was decreased by chronic alcohol

consumption, polyunsaturated fatty acid (PUFA), and conjugated linoleic acid

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(CLA) (Ntambi et al., 2004). Moreover, hormones that control metabolism, such

as INS, glucagon, and steroids, among others, also affect SCD1 expression

(Table 2). In addition, SCD1 is a central target for growth factors and hormones

that regulate key cell cycle events. A large number of potent mitogens (PDGF,

EGF, INS, FGF-2, FGF-4, KGF, TGF-, among other) have been shown to

stimulate SCD1 expression in a variety of untransformed human cell types

(Demoulin et al., 2004).

Table 1. Regulation of SCD1 by dietary factors.

(Adapted from Ntambi et al., 2003).

Dietary factors SCD1

Glucose ↑

Fructose ↑

Vitamin A ↑

Cholesterol ↑

PUFA ↓

CLA ↓

Alcohol ↓

Several common cell signaling pathways are activated upon growth factor

receptor stimulation by cytokines like EGF or INS, particularly Mitogen Activated

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Protein Kinase (MAPK) and Phosphosphatidylinositol 3-kinase (PI3K)/Akt

pathways. Demoulin et al., 2004 demonstrated that SCD1 was one of the most

regulated enzymes by mitogens, showing that the desaturase expression

increases upon cytokine stimulation in normal human fibroblasts. These authors

were able to demonstrate that growth factors activated sterol regulatory binding

protein-1(SREBP-1), a central transcriptional factor that activates lipogenic

enzymes, in PI3K/Akt -dependent manner, leading to up-regulation SCD1.

However, it is not known whether SCD5, the second human desaturase isoform,

is subjected to a similar regulation.

Table 2. Regulation of SCD1 by hormones. (Adopted from Ntambi et al., 2004).

Hormones SCD1

Insulin ↑

Growth hormones ↑

Estrogen ↑

Androgen ↑

Leptin ↓

Glugacon ↓

Thyroid hormone ↓

Dehydroepiandrosterone ↓

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Role of SCD in the regulation of lipid synthesis

SCD1 is not only a crucial controller of fatty acid composition but it also

plays a major role in the regulation of lipid synthesis. Hulver et al., 2005 showed

that an increase in SCD1 in human myoblasts was sufficient to increase the

formation of phospholipids and triacylglycerols. Conversely, inhibition of SCD1

activity in cancer cells reduced the de novo synthesis of phospholipids, including

the main membrane polar lipids phosphatidylcholine and

phosphatidylethanolamine (Scaglia, et al., 2005b; 2008; 2009). Data showing

that SCD1 activity also governs the rate of biosynthesis of triacylglycerol and

cholesterolesters in human cells and mouse tissues (Scaglia,et al., 2005b; 2008;

Ntambi et al., 2003) suggest that SCD1 is an overall regulator of the lipogenic

program in mammalian cells. Because of its multiple effects on cell metabolism

and signaling, SCD1 activity has significant implications on continuous replication

and survival of cells. The essential role of SCD1 in cell growth and proliferation

was demonstrated by work done both in human cells (Scaglia et al., 2005a:b;

2008; 2009) and in yeast (Nguyen et al., 2011) in which suppression of SCD1 by

genetic and pharmacological means promoted a slower rate of cell proliferation

and decreased survival.

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Implication of Stearoyl-CoA desaturases in chronic diseases

Studies in rodent models, specifically SCD1 knockout (abJ/abJ) and obese

(ob/ob) mice, have suggested that SCD1 plays an important regulatory and

function in chronic diseases; however, it is uncertain whether these findings will

be reflected in humans. Miyazaki et al., 2006: 2009 reported that abJ/abJ mice

exhibit resistant to diet-induced weight gains, increased energy expenditure,

increased INS sensitivity, and reduced TAG deposit in adipose tissues in relative

to control mice with SCD1. Another rodent model, famous ob/ob mice, further

supports major involvement SCD1 in chronic disease. In 2002, Cohen et al.,

2002 reported that ob/ob mice have strikingly high increased in SCD1 activity

(approximately 700%) in livers of leptin untreated mice verse SCD1 activity of

leptin treated ob/ob mice. Furthermore, these authors crossbred ob/ob mice with

abJ/abJ to evaluate the degree of SCD1 plays in development of obesity in leptin

deficient mice (Cohen et al., 2002). Interestingly, these double mutated (abJ/abJ;

ob/ob) mice showed remarkably reduced body weight without compromised lean

mass weight at all ages relative to controls (ob/ob) and demonstrated that SCD1

is required for ob/ob mice to fully develop its metabolic syndromes of obesity

(Cohen et al., 2002).

Frequent observations of an abnormally high presence of MUFA in cancer

cells lead many researchers to speculate involvement of SCD in cancers;

however, the actual amount of research data that is dedicated to elucidatingthe

link between cancer and SCD1 are few. Results of Scaglia et al., 2005a:b

suggested that upregulation of SCD1 in development of neoplastic cell

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transformation potentially be a necessary step involves in development of cancer,

and that elevated activity and expression of SCD1 provides a protective function

to minimize SFA-mediated apoptosis in lung cancer cells. In line with these

findings, various research groups have observed that inhibition of SCD1 lead to

impaired cell survival in human lung cancer cell lines (Morgen-Lappe et al., 2007,

Scaglia et al., 2008, Scaglia et al., 2009) and decreased prostate cancer cell

progression in mice (Fritz et al., 2010). In contrast to these findings, Moore et al.,

2007 have observed downreguation of SCD1 expression in human prostate

cancer tissues. In addition, unlike lung cancer cells, inhibition of SCD1in the

human breast cancer cells MDA-MB-468 did not affect cell death (Morgen-Lappe

et al., 2007). Interestingly, there is no single reported link of SCD5 in human

diseases. Consequently, it is clear that further investigations are needed to

elucidate the function of SCD1 in cancer and speculate about the possible roles

SCD5 isoform plays in disease.

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RESEARCH AIMS

Aim 1: To determine the levels of human stearoyl-CoA desaturase 5 protein

in human normal and cancer cells.

SCD1, the most characterized isoform of SCDs, is ubiquituously present in

most cells and tissues of mammalian organisms, including humans. This enzyme

is regulated by a number of nutrient and growth factors and is found

overexpressed in experimental and human cancers. Humans also expressed a

second SCD variant, the recently discovered SCD5, which was detected at the

mRNA level predominantly in pancreas and in fetal and adult brains. Unlike

SCD1, the functional role and regulation of SCD5 in human cells and tissues is

entirely unknown. The specific aim is to characterize for the first time the

presence of SCD5 protein across different human cell lines. In this aim, normal

lung fibroblasts (WI-38), simian virus-40 transformed lung fibroblast (SV40-WI38),

lung cancer cell lines (H460 and H1299 cells), as well as brain cancer U-87 cells,

will be used to determine SCD5 protein, in parallel to protein expression of SCD1.

Protein will be assessed using Western Blot technique. We hypothesize that the

SCD5 protein will reflect similarly to SCD1protein within the various cell types

across human cell lines.

Aim 2: To examine the regulation of human SCD5 in response to growth

factor stimulation.

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The nutritional and hormonal regulation of murine SCD isoforms and, less

extensively, human SCD1 has been characterized, but the modulation of human

SCD5 remains largely understudied. Previous studies reported that several

growth factors including INS and EGF, among others, upregulate SCD1 at both

mRNA and protein (Demoulin et al., 2004; Chang et al., 2005; Samuel et al.,

2001). Given that both SCD1 and SCD5 catalyze a similar reaction in human

cells (Wang et al., 2005), we hypothesize that SCD5 will be upregulated in

response to INS and EGF stimulation in a similar manner as SCD1. To test this,

INS, EGF, or both growth factors will be used to stimulate cells. Additionally,

because these growth factors are known to target the regulation of lipogenic

enzymes through the activation of the phosphatidylinositol 3-kinase (PI3K)/Akt

and ERK (MAPK) pathways, the levels of SCD5 and SCD1 will be determined

after blocking these signaling cascades with specific inhibitors.

Aim 3: To determine SCD5 protein in differentiated neuronal cells

The critical role of retinoic acid in neuronal differentiation has been long

established (Jones-Villeneuve et al., 1982); however, the involvement of MUFA,

particularly oleic acidin the regulation of neuronal differentiation has drawn

attention more recently (Tabernero et al., 2001). Studies performed in rat models

suggest a role for SCD1 in neuron growth and maturation (Polo-Hernández et al.,

2010). Although SCD5 is abundantly expressed in the brain, the implication of

SCD5 in the mechanisms of brain cell proliferation and differentiation has yet to

be elucidated. We hypothesize that SCD5 will be specifically modulated in

19

neurons in order to support the mechanisms of differentiation. In order to test our

hypothesis, we will use human neuroblastoma SH-SY5Y cell line, as well as

mouse Neuro2A cells, to study the regulation of SCD5 protein levels upon

induction of differentiation with retinoic acid.

20

MATERIALS AND METHODS

Chemicals and Supplies

Unless otherwise stated, all chemicals and supplies were purchased from

Sigma-Aldrich (St. Louis, MO) and Fisher Scientific (Pittsburgh, PA).

Cell culture

Normal human lung WI-38 fibroblasts, and its transformed strain, the

simian virus 40 transformed-WI 38 (SV40-WI38), WS-1 human skin fibroblasts,

H460 human lung cancer cells, H1299 human lung cancer cells, U-87 human

glioma cells, and SH-SY5Y human neuroblastoma were grown in Dulbecco’s

Modified Eagle’s Medium (DMEM). SCD5-overexpressing model in mouse

Neuro2A cells were recently generated by R. Ariel Igal. Briefly, mouse N2A

neuroblastoma cells were transfected with a protein expression vector (pCDNA4)

containing the human SCD5 gene and a gene encoding resistance to puromycin.

Control cells were transfected with empty plasmid alone. Cells were grown in

media containing puromycin, allowing for the selection of cells positively

transfected with SCD5 gene or plasmid control.

All cell lines were maintained in medium supplemented with 10% heat-

inactivated fetal bovine serum (FBS), 1% non-essential amino acid, 1% non-

essential vitamins, and 1% penicillin-streptomycin, at 37°C, 5% CO2 and 100%

humidity. All treatments were given to cells when cells have reached at least 80 %

confluency unless otherwise stated.

21

Analysis of SCD5 across human cell lines

For the detection of SCD5 across cell types and cell lines experiment,

WI38, SV40-WI38, H460, H1299, and U-87 cells were grown and maintained in

100 mm culture plates. Once cells have grown to target confluency, a fresh

complete cell medium was provided for 24 hours prior to harvest. Additionally, to

determine regulation of SCD5 in serum starvation induced quiescent state, WI38,

SV40-WI38, H460, and H1299 cells were cultured and maintained in100 mm

plates with a complete medium prior to treating cells with serum free medium.

Briefly, cells were rinsed twice with PBS and incubated 24 hours with a cell

medium (DMEM) supplemented with all the following except FBS: 1% non-

essential amino acid, 1% non-essential vitamins, and 1% penicillin-streptomycin.

After treatment, cells lysates were obtained as described below and stored for

further analysis.

Growth factor stimulation

To determine the effect of INS or EGF on regulation of SCD5, WI38,

SV40-WI38, H460, and H1299 cells were seeded and cultured in 60 mm culture

plates until cells have reached 80% confluency. Cells were then stimulated for 24

hours with 10 µg/mL INS, 100 ηg/mL EGF, or combination of both INS and EGF.

Additionally, groups of cells with PI3K inhibitor Ly 294002 and the MEK inhibitor

U0126, both at 20 µM final concentration. Control cells were treated with the

inhibitors vehicle, DMSO, at 0.1% (volume/volume) final concentration.

22

Induction of neuronal differentiation in SH-SY5Y using RA treatment

Regardless of a well-established method to use retinoic acid (RA) to

induce neuronal differentiation, discovery of the involvement of MUFA,

specifically oleic acid, in regulation of neuronal differentiation as a neurotropic

factor is relatively new (Tabernero et al., 2001). Human neuroblastoma cell line

SH-SY5Y was established originally from a parental cell line SK-N-SH in which it

was isolated from bone marrow biopsy of a four year old female neuroblastoma

patient (Ross et al., 1983). Conveniently, once SH-SY5Y cells undergo induced

differentiation using RA, dramatic differences in cell morphologies (dendrite

projection) and up-regulation of genes (GAP-43 and MAP-2) that are involved in

dendrite formations have been observed (Tabernero et al., 2001: 2002). For

these reasons, RA induced neuronal differentiation in SH-SY5Y provides a

valuable in-vitro model to study role of SCD5 in neuronal differentiation.

For the neuronal differentiation experiments, undifferenciated human

neuroblastoma SH-SY5Y cells were seeded and maintained in 12 well plates

until cells were treated at 50% confluency. Once cells have reached the desired

target confluency, cells were treated with 0.1% (volume/volume) vehicle (DMSO)

or inducer all- trans retinoic acid (RA) final concentration 20 µM for 76 hours. The

microscopic views of vehicle or RA treated cells were taken at the end of 96

hours prior to cells lysates preparation. All treatments were performed in

triplicates (n=3).

Cell Lysate Preparation

23

Cells were homogenized in 25 mM Tris-HCl pH 7.4, 1 mM EDTA, 0.1% SDS

plus a protease inhibitor cocktail, by brief sonication on ice. A portion of cell

lysate was saved for determination of protein concentration and the rest was

boiled for 5 minutes at 95 ˚C on a hot plate with 6X loading buffer ( 125 mM Tris-

HCl at pH 6.98, 2% SDS, 20% glycerol, 0.2% Bromophenol Blue, 5% β-

mercaptolethanol). Samples were stored in -80˚C until ready to use for

immunoblot determinations.

Determination of protein concentration

Total protein cell lysates was determined by using Bradford method

following the manufacturer’s instructions. Bovine Serum Albumin (BSA) was used

as standard. Optical density values of samples were determined in a microplate

reader (Molecular Devices, Sunnyvale, CA) at 595nm absorbance. The standard

curve regression was calculated using the SpectraMax software (Molecular

Devices), and only with standard curve slope that are greater than 0.94 were

used for calculation.

Immunoblotting

Twenty five to 100 g of proteins from lysates was loaded to a freshly

prepared 10% SDS-Polyacrylamide gel. Proteins were separated by

electrophoresis at 180V for 3 hours using an Owl electrophoresis system

(Thermo Scientific) coupled with an internal water cooling system temperature

set to 10˚C during the run. The buffer used in the electrophoresis procedure was

24

25 mM Tris, pH 6.8, 192 mM glycine, and 0.1% SDS. After the electrophoresis,

gels were transferred onto 0.45 µm nitrocellulose membranes (Bio-Rad, Hercules,

CA) at 300 mA for 18 hours at 4°C using a transfer system (Owl Separation

Systems) with buffer containing 25 mM Tris, 192 mM glycine, 20% methanol. The

nitrocellulose membranes were then blocked with 2 % non-fat dried milk power

(NFM) in PBS with Tween-20 (PBST:137 mM NaCl, 10 mM Phosphate, 2.7 mM

KCl, 0.002% Tween-20, adjusted pH to 7.0) for 1 hour at room temperature.

Membranes were washed three times with PBST 1X buffer and were then

incubated with appropriate primary antibodies (anti-SCD5, anti-SCD1, anti-FAS,

or anti-β actin as loading control). Antibodies raised against human SCD5, and

human SCD1 rabbit were generous gifts of Brent Rupnow (Bristol-Myers Squibb),

and Jean-Baptiste Demoulin (Université Catholique de Louvain, Belgium),

respectively. All other antibodies were purchased from Cell Signaling Technology,

Inc. (Danvers, MA). Each membranes were incubated with anti-SCD5 (1:500

dilution, in 2% NFM in PBST 1X) for 16 hours at 4˚C with slow rocking, anti-

SCD1 (1:5,000 in 2% NFM in PBST) for 1hour at room temperature, or anti-β

actin 1:4,000 in 2% NFM in PBST 1 hour at room temperature. Membranes

were then rinsed with PBST 1X for total of 3 times (10 minutes intervals) to

remove excess antibodies from the membrane, followed by incubation for 1 hour

at room temperature with appropriate secondary antibody (horseradish

peroxidase-linked anti-rabbit antibodies (Cell Signaling Technology). For

detection of SCD1 and SCD5, the secondary antibodies, goat anti-rabbit at a

1:3,000 dilution or anti-mouse at a 1:4,000 dilution were used. Membranes were

25

rinsed again with PBST 1X for total of 3 times (10 minutes interval) prior to

incubation with a chemiluminiscent reagent that detects peroxidase activity

(SuperSignal West Femto Maximum Sensitivity Substrate, Pierce, Rockford, IL)

for 5 minutes at room temperature. The membranes were exposed to Blue

Autoradiography film (BioExpress, Kaysville, UT) at various time points and

developed with a film processor. Films were scanned using a densitometry and

densities of bands were measured using Quanty One® software (Bio-Rad). In

some cases, chemiluminiscent bands were photographed and analyzed in a

ChemiDoc (Bio-Rad) digital image system. Protein band densities were

normalized to the β-actin content of the same samples with Quanty One®

software (Bio-Rad).

26

RESULTS

SCD5 is ubiquitously expressed across human cell lines

Every human tissue contains SCD1. SCD5 mRNA expression is found in

very low levels in most tissues with the exception of brain, where the levels of

SCD5 are particularly high (Zhang et al., 1999, Wang et al., 2005). However, the

levels of SCD5 protein and activity have never been assessed in human cells

and tissues. Increased SCD1 are associated with various types of cancers in

humans (Lu et al., 1997, Thai et al., 2001, Igal 2010), whereas there is no known

association of SCD5 with disease in humans. To initiate the path toward

determining the functional role of SCD5 and its potential association with

diseases, we characterized the basal level of SCD5 protein in a series of normal

and cancerous human cells: lung cancer cell lines (H460, and H1299), a glioma

cell line (U-87), as well as normal lung fibroblast and simian virus 40-transformed

lung fibroblast cell line (SV40-WI38). Brent Rupnow (Bristol-Myers Squibb)

generated the first specific antibody against human SCD5 protein. In order to

validate this antibodies in our experiments, we generated an SCD5-

overexpressing cell model in mouse Neuro2A cells, which does not express

SCD5, by stable transfection of a pcDNA4 plasmid encoding the human SCD5

cDNA. Controls cells were transfected with empty plasmid. After selection of

SCD5-containing clones by resistance to puromycin, cell lysates were analyzed

for the expression of SCD5 protein. As shown in Figure 4, the antibody

recognizes a protein in SCD5-overexpressing cells with the expected ~31KDa

27

molecular weight for SCD, whereas control cells do not show expression of this

protein. This study suggests that our antibody is able to specifically detect SCD5

in Western blot analysis.

Figure 4. Verification of anti-SCD5 antibody efficacy. Mouse neuroblastoma Neuro2A cells were stably transfected with human SCD5 expression vector pcDNA4-SCD5 or a control vector pcDNA. Total cell lysate was prepared and recombinant SCD5 protein was detected using anti-SCD5 antibody in Western Blot as described in the method.

For our next experiment in which we wished to determine the levels of

both SCD1 and SCD5 in human cells, normal and neoplastic cells were

SCD5

Co

ntr

ol (

pc

DN

A4 )

pcD

NA

4-S

CD

5

β-actin

28

incubated in 10%FBS DMEM for 24h and then cell were harvest and prepared for

immunoblot (Figure 5). SCD5 protein was ubiquitously expressed among all cell

lines, whereas SCD1 protein was highly expressed in cancerous cell lines as

expected. Virtually no SCD1 protein band was detected in normal lung WI38

fibroblasts, and a relatively small SCD1 protein band was detected in SV40-WI38

cell line compared to SCD1 protein bands in lung cancer and glioma cell lines.

When sera were removed for 24 h from the media of WI-38, SV40-WI38, and two

lung cancer cell lines (H460, and H1299) prior to harvest, SCD1 protein band

appeared in WI-38 cells as shown in Figure 5B, but it the SCD1 protein band

was nowhere near the level of SCD1 bands in lung cancer cells (Figure 5B).

Overall, SCD5 protein was ubiquitously present among normal to transformed

lung fibroblast cells as well as in lung cancer cells and glioma cells. As a matter

of fact, SCD5 appears to be the only SCD isoform expressed in normal human

cells. The highest level of SCD5 protein band was detected in glioma (U-87), a

finding that confirms reports of highest mRNA level of SCD5 in brain tissue

(Zhang et al., 1999, Wang et al., 2005).

29

A B

Figure 5. SCD5 protein is ubiquitously expressed in various human cell lines. (A) Western blot analysis for SCD5, SCD1, or β-actin as loading control from the following cell lines after 24 hours treatment with complete medium: Human normal fibroblast, WI38; simian virus 40-transformed-WI38, SV40-WI38; H460 and H1299 human lung cancer cell lines, and U-87 human glioma cancer cells. (B) Western blot analysis of SCD5, SCD1, or β-actin as loading control in cell lines incubated for 24 hours treatment with serum-free medium A total of 100 µg of protein was loaded per well. * Unidentified protein.

Complete medium Serum-Free medium

30

SCD5 protein is sensitive to INS but not to EGF stimulation

Human SCD1 is upregulated by growth factors that activate the cell cycle,

such as INS, EGF, PDGF, among others (Demoulin et al., 2004). The response

of SCD5 to these cytokines has not been studied to date. To gain insight into the

regulation of SCD5 protein by growth factors, we stimulated normal and lung

cancer cells with INS, EGF, or both for 24h prior to harvest and analyzed the

levels of proteins by Western blotting with specific antibodies. Overall, INS

appears to elevate the levels of SCD5 protein in all cells, except H460 cells in

which SCD5 protein was not modified by any treatment (Figure 6A-D). The most

pronounced effect on SCD5 content was observed in H1299 cells upon INS

stimulation. In these cells EGF was unable to affect SCD5 levels (Figure 6C).

SCD1 protein, on the other hand, seems to be increased among all cells with INS

as well, except in normal fibroblasts in which no SCD1 protein level was detected.

As previously mentioned, growth factors like INS and EGF induce the

transcription of lipogenic enzymes by activating critical signaling cascades such

as PI3K/Akt and ERK (MAPK). Hence, we subjected the cells to stimulation with

the growth factors in presence of a MEK inhibitor that blocks ERK activation

(U0126), or a PI3K inhibitor (Ly) that blocks the activation of Akt signals. Overall,

these inhibitors showed mixed effects on the levels of SCD1 and SCD5 proteins

in different cells. In WI38 cells, decreased SCD5 protein was observed in cells

treated with PI3K inhibitor in growth combination of both growth factors (INS and

EGF). In addition, SCD5 protein was decreased in SV40-WI38 cells stimulated

with EGF and incubated with the PI3K inhibitor whereas both inhibitors promoted

31

a slight decrease in SCD1 protein (Figure 6B). In H1299 cells, neither PI3K nor

MEK inhibitors were effective in regulating either desaturase. Unlike H1299 cells,

SCD1 of H460 cells was reduced by treatments with both PI3K and MEK

inhibitors (Figure 6D).

Altogether, similar to SCD1, it appears that SCD5 protein is globally

sensitive to INS stimulation whereas unlike SCD1, EGF does not have a strong

activating effect in SCD5. In addition, it appears that SCD1 insulin-mediated

induction of SCD5 is controlled by the PI3K/Akt signaling mechanism.

32

A B

C D

Figure 6. Effect of growth factors on the expression of SCD1 and SCD5 across cell types and cell lines. (A) Human normal lung fibroblast (WI38) (B) Oncogene transformed normal lung fibroblast ( SV40-WI38) (C) Human lung cancer cells (H1299) or (D) Human Lung cancer cells (H460) were treated with insulin (INS), EGF, combination of both , plus or minus 20 µM Ly294002 (Ly, PI3K inhibitor) or U0126 (U0, MEK inhibitor). SCD1, SCD5 and β-actin were detected by Western Blot. A total of 25 µg of protein was loaded per well.

Simian virus 40 transformed- WI38

SV40-WI38

Lung cancer H1299 cells

Normal fibroblast

WI-38

Lung cancer H460 cells

33

Regulation of SCD5 during neuronal differentiation

SCD5 is abundantly expressed in brain in adult humans, however in fetal

brains SCD5 is the predominant form of Δ9 desaturase in which, it is responsible

for biosynthesis of MUFA, specifically oleic acid (Wang et al., 2005). It was not

until recently that oleic acid is being studied for its positive role in neuronal

differentiation (Tabernero et al., 2001). However, the regulation, and potential

functional role, of SCD5 in neuronal differentiation is still unknown. Here, we

investigated whether SCD5 levels were affected by the process of neuronal

differentiation in SH-SY5Y neuroblastoma cells. In this cell model, neuronal

differentiation was induced using retinoic acid (RA) for 4 days, and SCD5 protein

expression was analyzed by Western blot with specific antibodies. Microscopic

observation showed a large difference between control (DMSO) and RA treated

groups after 4 days of treatment, in which RA induced remarkable axonal growth

(Figure 7A) accompanied by cell flattening, tow typical markers of differentiation.

Western blot analyses showed a slight increase in SCD5 protein expression in

cells undergoing neuronal differentiation (Figure 7B-C). These results indicate

that SCD5 is somewhat responsive to the mechanism that trigger neuronal

differentiation, but additional studies are needed to determine whether these

slight changes play a role in this process.

34

C

0

0.2

0.4

0.6

0.8

1

1.2

Control RA

SCD

5/β

-act

in (A

rbit

rary

val

ue

)

A B

C

Figure 7. Effect of RA induction in neuronal differentiation on SCD5. (A) A representative microscopic view of SH-SY5Y cells 4 days after treated with control (0.1% vehicle, DMSO) or all trans retinoic acid (RA, 20µM). (B) Western Blot analysis of SH-SY5Y cells treated with 0.1% vehicle (DMSO) or all-trans retinoic acid (20µM) for 96 hours. Cells were lysed and subjected to Western Blot analysis for SCD5, and β-actin. (C) Densitometry analysis of Western blot determinations of SCD5 in undifferentiated and differentiated neurons. SCD5 values were normalized against β-actin as loading control. Data represent the means of three independent determinations ± SE. Effect of RA in induction in neuronal differentiation on SCD5 were not significantly different (*P>0.05) compared with controls of triplicate experiments.

Control RA

*

34

DISCUSSION Dysfunctional activity and abnormal expression of SCD1 is widely linked to

several chronic diseases including obesity, diabetes, and cancers. In mammalian

cells, SCD1 serves as a critical key controller and regulator of fatty acid

composition and lipid synthesis (Hulver et al., 2005, Scaglia et al.,

2005b:2008:2009). Humans have two SCDs, namely SCD1 and SCD5, and its

amino acid sequence against all four of mouse SCD isoforms have revealed that

mice lack SCD5. Compared with SCD1, tissue specific expression of SCD5 have

been reported in brains of adult embryo in humans (Wang et al., 2005), brains of

bovines, sheep, and pancreas and brains of chickens (Lengi et al., 2008). SCD5

is a functional ∆9 desaturase, capable of catalyzing its enzymatic activity to

biosynthesize MUFA from SFA (Wang et al., 2005). Few studies have

characterized gene expression of SCD5 in normal human tissues (Zhang et al.,

1999, Beiraghi et al., 2003, Wang et al., 2005), and regulation of human SCD5 is

virtually unknown.

In the present study, we attempted to initiate characterization of basal

SCD5 protein level and its regulation by growth factors (INS, EGF, or both) in

human normal and cancer cells. Assessment of basal SCD1 and SCD5 protein

revealed that SCD5 protein is rather abundantly expressed across normal to

neoplastic cells in humans. In our lab, Scaglia et al., 2005a have reported that

SCD1 protein in SV40-WI38 cells is ~3-fold higher than its parental normal WI38

cell line. In this report, we also observed that SCD1 protein is increased in SV40-

35

WI38 relative to WI38 cell line. Moreover, we observed remarkably high levels of

SCD1 protein expressions in cancer cells in comparison to WI38 cells.

Several studies consistently reported that SCD1 is under positive control

of INS and EGF (Enser 1979, Ntambi et al., 1999, Bene et al., 2001, Zhang et al.,

2001, Lefevre et al., 2001, Legrand et al., 1991, Samuel et al., 2001, Demoulin et

al., 2004, Chang et al., 2005). The mechanism in which INS or EGF attributes to

control and regulates transcription factors are mediated through phosphorylation

and activation of INS or EGF specific cell receptors, namely insulin receptor

tyrosine kinase (IRK) or epidermal growth factor receptor (EGFR). Upon

activation of IRK by INS, a subunit (p85) of PI3K is recruited as an adaptor

protein, thereby activating PI3K (Alessi et al., 1998, Yenush et al., 1998).

Studies reported that PI3K is a major pathway in which INS modulates gene

transcription (Band et al., 1997, Dickens et al., 1998, Sutherland et al.,

1995:1998). Similarly, EGFR activation initiates recruitment of specific adaptor

proteins lead to activate MAPK (Shultz 2007). Among many difference, INS is

the main regulator of SREBP-1 and EGF is not (Demoulin et al., 2004). SCD1 is

known to be regulated under control of SREBP (Ntambi et al., 2004, Demoulin et

al., 2004). In the present study, we hypothesized that because SCD1 is

upregulated in response to INS and EGF stimulations, SCD5 is likely to be

upregulated in similar manner as of SCD1. Using normal to cancerous cells, we

observed that SCD5 protein was unresponsive to EGF stimulation across cell

lines. Unlike our hypothesis, SCD5 did not respond in a similar manner to SCD1,

which suggests that human SCD5 is regulated differently than SCD1; however,

36

more studies need to be conducted to investigate its distinctive regulation. In

addition to investigation of SCD regulation by growth factor stimulations, we

investigated effects of MEK inhibitor and PI3K inhibitor in normal to cancerous

cells. Our observation of mixed effects of MEK inhibitor and PI3K inhibitor on

SCD1 and SCD5 suggest that these may have been resulted of cell-type specific

response to inhibitors. It will be interesting to test effects of these inhibitors on a

large panel of tissue specific cancerous cells with known gene mutations in

regulation of SCD5. Overall, our data suggests that activation of PI3K/Akt

pathways by INS may partially regulate human SCD5.

Our last research aim was to investigate the potential role of SCD5 during

neuronal differentiation. In human embryonic brains, SCD5 is the main form of

∆9 desaturase, whereas adult brain tissues contain approximately equal gene

expression of SCD5 to SCD1 (Wang et al., 2005). Although previous studies

consistently reported that oleic acid (18:1) acts as a neutrophic factor that is able

to initiate neuronal differentiation (Tabenero et al., 2001, Rodríguez-Rodríguez et

al., 2002, Meldina et al., 2002, Velasoo et al., 2003, Granda et al., 2003, Polo-

Hernández et al., 2010), it is only in recent years that the positive role of SCD1 in

neuronal differentiation hasbeen investigated using the rat animal model (Polo-

Hernández et al., 2010). Interestingly, SCD2 isoform is the main ∆9 desaturase

catalyzing the production of oleic acid in brain of mouse and rat (Kaestner et al.,

1989, Miyazaki et al., 2004, Garbay et al., 2000). Granted that rodent SCD2

isoform shares similar tissue distribution with human SCD5, we anticipated the

human SCD5 protein involvement in neuronal differentiation. In spite of our

37

anticipation and distinguishable axonal growth of cells undergoing neuronal

differentiation, we observed only a slightly increased in SCD5 protein in these

cells in relative to control cells. In addition, SCD1 protein was undetectable

among these cells. We suspect that SCD1 protein bands will likely to reappear

once we starve cells by removing serum.

At this point, it is unclear as to why WI38 and neuroblastoma SH-SY5Y

cells are expressing SCD5 protein under regular growth condition which includes

serum. From our experiment data, we now know that SCD5 is partially regulated

by activation of PI3K/Akt pathway, and EGF stimulation does not have impact on

SCD5. Since all the cell lines we have test contains wild-type EGFR (WI38,

SV40-WI38, H1299, and H460), it is attempting to investigate whether SCD5

insensitively in response to EGF stimulation holds true for cells with EGFR

mutation. Another interesting project will be testing is the role of SCD5 in cell

cycle and apoptosis. Among other important role of SCD, Scaglia et al., 2005b

demonstrated that SCD1 acts as a protector role in avoidance of apoptosis,

mainly to avoid SFA-mediated lipotoxicity which leads to cell death. In order to

prove the hypothesis, Scaglia et al., 2005b generated SV40-WI38 cells deficient

in SCD1 using pcDNA3-human SCD1 antisense and transfected cells. Using

stably knockdown SCD1 deficient cell model, palmitic acid (16:0) induced

apoptosis.

In conclusion, the data presented here show for first time that the human

SCD5 protein is abundantly expressed in normal to neoplastic cells, and that

regulation of SCD5 protein by INS is likely by activation of PI3K/Akt pathway.

38

These finding is a step closer toward understanding implication and regulation of

human SCD5 protein in normal and cancerous cells. Besides our initial finding of

abundantly expressed human SCD5 protein across normal and cancerous cells,

we found that INS regulated SCD5, likely through the activation of the PI3K/Akt

pathway. In summary, our findings implicate that the modulation of SCD5 is by

critical factors that control cell growth and survival in human cells. Taken together,

these data suggests that regulation of human SCD5 could be a key to finding

new therapeutic interventions.

39

CONCLUSIONS

Our experiments resulted the following conclusions.

1. In human normal, transformed, and cancer cells, SCD5 protein is

ubiquitously expressed. SCD1 protein was significantly elevated in cancer

cells and transformed cells whereas normal cells exhibit extremely low

levesl of this SCD isoform. On the other hand, SCD5 appears to be the

dominant SCD variant in normal fibroblasts. SCD5 was detected in cancer

cells but, unlike SCD1, its level does not seem to increase with the

neoplastic transformation process. Importantly, U-87 neuroblastoma cells

exhibit great level of SCD5 suggesting a potential function of SCD5 in

brain cells.

2. Similar to SCD1, SCD5 protein levels are upregulated by INS, likely

through the PI3K/Akt. Unlike other research groups’ findings of regulation

of SCD1 by both INS and EGF, we report that SCD5 protein is not

sensitive to EGF-mediated regulation. This finding suggests that

induction of cell proliferation by this growth factor may not include the

participation of SCD5 in this process.

3. In retinoic acid-induced neuronal differentiation, SCD5 is slightly increased.

The precise implication of SCD5 in this mechanism is not clear and

requires further investigation.

40

FUTURE WORK

In the presented study, we only detected SCD5 protein using antibodies

and this does not mean those proteins were serving functionally to synthesize

oleic acid. Because of that reason, fatty acid composition, specifically

MUFA/SFA, assay will provide as good support of a new hypothesis. In order

study functional role of SCD5, the next phase of the study will involve generating

various mutant cells. Possible mutant cells in construct I have in mind are the

ones with different placement of histidine motif box in SCD5 protein amino acid

sequences.

For the future studies, I would chose various methods in order to

investigate role of SCD5 protein in neuronal differentiation. These may include a)

optimization of serum and RA concentration b) determining SCD activity by

measuring MUFA/SF throughout all states of treatments c) utilize other known

nerve derived growth factors. Ultimately, neuroblastoma cells with knockout

SCD5 or overexpression SCD5 can provide as a great tool to further study the

role of SCD5 in neuronal differentiation. Currently, the lab is investigating the

function of SCD5 in neuronal cells using stable mouse neuron cells exogenously

expressing the human SCD5 protein. Additionally, it will be important to use RT-

PCR to detect mRNA expression of SCD5 in parallel with SCD1 across different

cell lines to support the studies done with SCD1 and SCD5 antibodies.

41

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