mesenchymal progenitor cells in intramuscular...

Mesenchymal progenitor cells in intramuscular connective tissue

development

Mesenchymal progenitor cells in intramuscular connective tissue

development

Min Du, Ph.D. Professor and Endowed Chair

Department of Animal SciencesWashington State University

Pullman, WA

Beef quality is mainly determined by marbling and tenderness.

Marbling is the primary criterion for grading beef carcasses.

Only carcasses with moderate to abundant marbling qualify for high quality grades.

Beef Quality: marbling and tenderness

Beef tenderness is determined by:

Myofibrillar effect: aging, stretching carcasses improves tenderness.

Background toughness: connective tissue, mainly collagen and its cross‐linking.

Both collagen content and cross‐linking increase as animals become older.

Question: How to reduce collagen content and cross‐linking in muscle?

Beef Quality: marbling and tenderness

Collagen content and cross‐linking, two critical control points:

Fibroblasts synthesize collagen and other fibrotic proteins in connective tissue.

Fibroblasts secrete enzymes catalyzing collagen cross‐linking.

Tenderness remains a top problem for beef

Collagen content

Collagen cross‐linking

How to reduce fibroblasts?

How to reduce fibroblasts?

Fibroblasts are derived from mesenchymal multipotent cells, primarily during the early developmental stage.

More importantly, fibroblasts and adipocytes share a common immediate progenitor cells, so called fibro/adipogenic cells (FAPs).

Tenderness remains a top problem for beef

: Why do some stem cells become myogenic cells, while others become fibro/adipogenic cells?

: Why do some progenitor cells become adipocytes, while others become fibrogenic cells?

Adipocytes

Fibroblasts

Satellite cells

Myocytes

Myogenic progenitor cells

Fibro/adipogenic progenitor cells

in fetuses

Stem cells in early embryos

Resident fibro/adipogenicprogenitor cells

In the stromal vascular fraction of mature muscle

1

2

1

2

The mechanisms regulating mesenchymal progenitor cell commitment to myogenesis, adipogenesis and fibrogenesis are poorly defined, especially in livestock.

Adipocytes

Fibroblasts

Satellite cells

Myocytes



In fetuses




1

2

Tumor necrosis factor (TGF) signaling pathway and Zfp423 transcription factor appear to have critical roles in determining progenitor commitments to either adipogenicor fibrogenic lineages.

Adipocytes

Fibroblasts

Satellite cells

Myocytes



In fetuses




1

2

Transforming growth factor (TGF)and fibrogenesis

TGF is a major signaling pathway promoting fibrogenesis.

TGF signaling is enhanced by inflammation and other factors.

Obesity is known to induce inflammation and TGFsignaling.

Fetal stage is critical for fibrogenesis.

AAAAA

Smad7CollagenCross‐linking enzymes

Smad 2

TGFRII

Kinase

TGFRI

Kinase

TGFRII

Kinase

TGFRI

Kinase

TGF

S465

S467

Smad 3

S422

S424

Smad 3

P

PP

Smad 3

P

PP

GST204

Objective: Thus, a maternal obesity sheep model was used to assess the role of TGF‐β signaling on fibrogenesis during early development.

Animals: Non‐pregnant ewes were assigned to a control diet (Con, fed 100% of NRCnutrient recommendations, n = 6) or obesogenicdiet (OB) fed 150% of NRC recommendations, n = 6) from 60 days before conception. Fetal semitendinosus (St) muscle was sampled at 135 days of gestation (term 148 days).

Methods: Histochemical analyses, Hydroxyproline assay, Real‐time PCR, Western blot analyses, Electrophoretic mobility shift assay (EMSA).

Experimental design

Huang et al., AJP‐ Endocrinology and Metabolism 298:1254‐1260, 2010

0

0.5

1

1.5

Con OB

Arb

itary

Uni

t *

Xu et al., Endocrinology, 2010, 151: 380.

Enhanced tumor necrosis factor (TNF)expression in OB fetal muscle

TNF: A marker of systemic inflammation.NF‐B: A major inflammatory signaling.

0.2

0.6

1

1.4

1.8 *

NF-B p65

OB

GAPDH

Con

00.5

11.5

22.5

33.5

Con OB

TGFβ

Prec

urso

r

Tubulin

TGFβ Precursor

*

Con OB Con OB


Transforming growth factor (TGF) content increased in

OB fetal muscle

AAAAA


Smad 2

TGFRII

Kinase

TGFRI

Kinase

TGFRII

Kinase

TGFRI

Kinase

TGF

S465

S467

Smad 3

S422

S424

Smad 3

P

PP

Smad 3

P

PP

GST204

Smad binding element (SBE)


Binding to Smad responsive element was increased in OB fetal muscle

Shift band

Free probe

Con OB

0

0.5

1

1.5

2

2.5

Con OB

*

Rel

ativ

e SB

E sh

ift

P32 labeled Smad binding

elements

0123456

Con OB

Smad

7

*P < 0.05


Expression of Smad7 was enhanced in OB

fetal muscle

AAAAA


Smad 2

TGFRII

Kinase

TGFRI

Kinase

TGFRII

Kinase

TGFRI

Kinase

TGF

S465

S467

Smad 3

S422

S424

Smad 3

P

PP

Smad 3

P

PP

GST204

0.0

1.0

2.0

3.0

4.0

Con OB

Fibr

onec

tin

*P < 0.05

00.5

11.5

22.5

33.5

Con OB

Proc

olla

gen

Type

I

&P = 0.08

Procollagen: Precursor of collagen.Fibronectin: Extracellular matrix glycoprotein.


Expression of TGF target genes was enhanced (Connective tissue content)

0

1

2

3

4

Con OB

LH2b

*P < 0.05

0123456

Con OB

Lysy

loxi

dase

*P < 0.05

00.5

11.5

22.5

Con OB

P4H

A

&P = 0.05

P4HA: prolyl 4‐hydroxylase, formation of hydroxyproline.LH2b: lysyl hydroxylase‐2b, collagen cross‐linking.Lysyl oxidase: amine oxidase, collagen cross‐linking.


Expression of TGF target genes was enhanced (Collagen cross‐linking)

40

80

120

160

Con OB

0

10

20

30

40

Con OB

Col

lage

n ar

ea/v

iew

ar

ea (%

)

*

Col

lage

n/dr

y m

uscl

e w

eigh

t (m

g/g)

CON

OB

x100 x400


*

Transforming growth factor (TGF)and fibrogenesis

Sheep fetal muscle at 135 dG when skeletal muscle matures (term day 148 gestation).

Con OB

Xu et al., Endocrinology, 2010, 151: 380.

It appears that the fibro/adipogenic pathway was enhanced in fetal muscle due to obesity and over‐nutrition in early development – Control point 1.

Adipocytes

Fibroblasts

Satellite cells

Myocytes



In fetuses




1

2

To further study mechanisms regulating lineage commitments of mesenchymal stem cells and progenitor cells, we used Wagyu and Angus cattle.

Adipocytes

Fibroblasts

Satellite cells

Myocytes



In fetuses




1

2

Wagyu cattle are known for its extremely high marbling

Wagyu cattle are known for their extremely high marbling.

Due to their similarities in growth characteristics, but sufficient difference in marbling, Wagyu and Angus cattle are frequently compared.

We sampled the Sternomandibularismuscle for analyses.

0.00.51.01.52.02.5

CEBPβ

P = 0.2864

Arb

itrar

y un

its

0.01.02.03.04.05.0

PPARγ

Arb

itrar

y un

its *

0.00.51.01.52.02.5

Zfp423

Arb

itrar

y un

its *

0.0

1.0

2.0

3.0

4.0

Intramuscular fat(IMF)

*

% o

f eth

er e

xtra

ct

Wagyu muscle have enhanced adipogenesis

0.0

1.0

2.0

3.0

4.0

CEBPα

*

Arb

itrar

y un

its

Duarte et al., J. Anim. Sci., 2013, 91: 2938

0.00.51.01.52.02.5

FGF-2

Arb

itrar

y un

its

*

0.00.51.01.52.0

FGFr

Arb

itrar

y un

its

FGFR

*

0.0

1.0

2.0

3.0

FIBRONECTIN

Arb

itrar

y un

its

Fibronectin

*

0.0

1.0

2.0

3.0

4.0

TGFβ

Arb

itrar

y un

its *

Wagyu cattle have higher fibrogenesis


0.0

1.0

2.0

3.0

4.0

COL I COL IIICol

lage

n m

RN

A ex

pres

sion

(arb

itrar

y un

its)

Collagen I Collagen III

*

*

0.0

1.0

2.0

3.0

4.0

Collagen content

*

mg/

g of

mus

cle

Wagyu cattle are higher collagen content


100x 200x 400x

Angus

Wagyu

Wagyu cattle are higher collagen content


Wagyu

Angus

0

50

100

150

20 45 70 95 120

Diameter (µm)

Freq

uenc

y

020406080

100

Breeds

*

Dia

met

er

(µm

)

Angus Wagyu


Wagyu have decreased myogenic pathway

Attenuated fetal myogenesis forms less muscle fibers.

The larger muscle fiber diameter in Wagyu despite less muscle mass shows less muscle fiber numbers, indicating attenuated myogenesis during early development.

Wagyu

Angus

It appears that both fibro/adipogenic pathway (1st question) and adipogenic differentiation (2nd question) are enhanced in Wagyu, which we are exploring.

Our current studies focus on Zfp423.

Adipocytes

Fibroblasts

Satellite cells

Myocytes



In fetuses




1

2

Zfp423 in adipogenesis of fibro/adipogenicprogenitor cells

Key question 2: what determines the lineage commitment of fibro/adipogenic cells (FAPs)?

Zinc finger protein (Zfp) 423 is a newly identified transcription factor regulating adipogeniccommitment of FAPs, which induces adipogenicdifferentiation and reduces fibrogenesis.

AdipocytesFAPs

Zfp423 PPAR

Pre‐Adipocytes

Adipogeniccommitment

Adipogenicdifferentiation

What is Zfp423?

We hypothesized that Zfp423 is critical for adipogenic differentiation of intramuscular fibro/adipogenic progenitor cells.

Zfp423 is correlated with enhanced adipogenesis and reduced fibrogenesis in beef cattle.

Animal:

The Sternocleidomastoid muscle was sampled from the carcass of an Angus heifer (20 months of age) immediately after slaughter.

Stromal vascular cells were separated, immortalized by over‐expression of telomerase, and cloned.

Three clones with high adipogenic and low adipogenicpotential respectively were selected.

Methods

0

0.5

1

1.5

2

2.5

Highadipogenic

Low adipogenic

*

High adipogenic

Low adipogenic

A

B

C

D

Arbitrary un

its


Huang et al., PLOS one, 2012, 7: e47496

0.0

1.0

2.0

3.0

4.0

5.0

Zfp423 PPARr C/EBPa C/EBPb Fibronectin TGFb CollagenI CollagenIII

0.0

3.0

6.0

9.0

Zfp423 PPARr C/EBPa C/EBPb Fibronectin TGFb CollagenI CollagenIII

Before differentiation

Adipogenic differentiation for 13 days

**

*

**

&&

& &

Arbitrary un

itsArbitrary un

its

C/EBPαPPARγ C/EBP

C/EBPαPPARγ C/EBP

TGF‐

TGF‐

mRNA expression of Zfp423 and other genes

Low adipogenicHigh adipogenic

*P < 0.05; &P < 0.10.


0

0.4

0.8

1.2

1.6

2

Low adipogenic High adipogenic

After adipogenic differentiationBefore adipogenic differentiation

&

0.0

1.0

2.0

3.0

4.0

Low adipogenic High adipogenic

*

High adipogenicLow adipogenic

TGF‐

Tubulin

High adipogenicLow adipogenic

TGF‐

Tubulin

Arbitrary un

its

Arbitrary un

its

TGF signaling was higher in Low Adipogenic Cells


0

0.5

1

1.5

2

2.5

3

3.5

4

Zfp423 C/EBPa C/EBPb PPARr Fibronectin TGFb

Adipogenic Non‐adipogenic

NA Zfp423 transfection NA GFP transfection

aa

bba abbc

ca aab b

a

a

bcc

ab b

ab ab b

ab

Arbitrary un

its

C/EBP C/EBP PPAR TGF‐

Zfp423 over‐expression in low adipogenic cells enhances their adipogenic differentiation

(Bars marked with different letter differ, n =3.)Huang et al., PLOS one, 2012, 7: e47496

TGF‐

Zfp423 over‐expression in low adipogenic cells enhances their adipogenic differentiation

(Bars marked with different letter differ, n =3.)


High adipogenic Low adipogenic Low adipogenic + Zfp423 Low adipogenic + eGFP


High adipogenicLow adipogenic High adipogenic + shZfp423

High adipogenic + GFP


Both high and low adipogenic cells are from the same bovine animal.

They have identical genetic composition.

The difference in their adipogenic and fibrogenicdifferentiation should be due to epigenetic modifications.

Because these cells have been immortalized and cloned, only stable epigenetic modifications, or DNA methylation, is expected to maintain.

Why does Zfp423 express at a higher level in high adipogenic cells?

0

1

2

3

4

5

TGF‐b Zfp423

Low adipogenic

High adipogenic

*

*

Arbitrary un

its

TGF‐

Zfp423 promotor contains rich GC sites, and higher methylation in low compared to high adipogenic cells

ATG

Methylated DNA immunoprecipitation (MeDIP) was used to measure the DNA methylation of Zfp423 promoter.



Why do TGF and Zfp423 expression differ between low and high adipogenic cells?

DNA methylation is a stable epigenetic modification which inhibits gene expression, determining cell phenotypes.

TGF DNA methylation was higher, while Zfp423 DNA methylation was lower in low adipogenic cells, consistent with higher adipogenic and low fibrogenic differentiation of FAPs.


What regulates Zfp423 expression?

We hypothesized that epigenetic modifications have key roles in regulating Zfp423 expression.

Epigenetic modifications are mainly referring to DNA methylation and histone modifications.

Histone modifications include histone methylation and acetylation, and others.

What regulates Zfp423 expression?

Polycomb repressive complex 2 (PGC2) catalyzes repressive histone modifications, H3K27me3.

Trithorax group proteins (TrxG) catalyze permissive histone modifications, H3K4me3.

Both H3K27me3 and H3K4me3 co‐exist in key developmental genes, forming “bivalent” status.

Lack of stimulation, repressive histone modifications convert to DNA methylation for permanent silencing.

Sect 1 Sect 2Sect 3 Sect 4

B

A

Yang et al., Diabetes, 2013, 62:3727

Zfp423 promoter has a “bivalent” status

Maternal obesity enhances Zfp423 expression in fetal tissue via inducing epigenetic changes

To test the role of epigenetic changes in the regulation of Zfp423 expression, we used a diet‐induced obesity pregnant mouse model.

Female mice were fed either a control diet (Con) or an obesogenic diet (OB) for two months to induce obesity.

Fetal mice at E14.5 were collected for analyses, when early adipose development has initiated.

Con

OB

Sec1 Sec 2 Sec3 Sec4

CON vs. OB: 30.0% vs. 16.9% 38.0% vs. 21.0% 4.4% vs. 2.1% 0.4% vs. 0.0%


DNA methylation was lower in Zfp423 of fetal tissue of obese mothers

0.00

2.00

4.00

6.00

8.00

10.00

Con OB

H3K27me3

Perc

ent o

f inp

ut (%

)

0.0

1.0

2.0

3.0

4.0

5.0

6.0

Con OB

Percen

t of inp

ut (%

)

H3K4me3

*

H3K27me3 H3K4me3

*

IPInput

IgG

Con OB

IgGInput

IP


Inhibitory histone modification, H3K27me3 is lower, and permissive modification, H3K4me3, is higher in OB


Inhibitory histone modification, H3K27me3 is lower, and permissive modification, H3K4me3, is higher in OB

0

0.2

0.4

0.6

0.8

1

1.2

Con HFD

*

Zfp4

23 p

rote

in c

onte

nt(A

rbitr

ary

Uni

t)

OB

Con OB

Zfp423

β-Actin

H3K27me3 Methylated CpG Unmethylated CpG

Normal pregnancy

PRC2

Zfp423 promoter

PRC2

Maternal obesity

PRC2PRC2

Zfp423 promoter

Acetylation

AC

ACACAC

Zfp423 Zfp423

Proposed mechanism linking maternal obesity to epigenetic modifications in the Zfp423 promoter

In summary, there are two major control points for reducing intramuscular fibrogenesis: Stem cells to either myogenic or FAP lineage. FAPs to either adipogenic or fibrogenic lineage.

Adipocytes

Fibroblasts

Satellite cells

Myocytes



In fetuses




1

2

Multipotentcells

Muscle cells

Adipocytes

Fibroblasts

Epigenetic changes in progenitor

cells

cell signaling pathways

Genetic, nutritional and other factors

Mechanisms regulating mesenchymalprogenitor cell differentiation

What controls mesenchymal progenitor cell differentiation?

Critical questions.

If we know these answers,

We can:

Improve lean/fat ratio and production efficiency.

Reduces connective deposition and increase tenderness of beef.

Enhance intramuscular adipogenesis and marbling.

Funding support:Funding support:

mesenchymal progenitor cells in intramuscular...

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