zonal induction of mixed lineage kinase zpk/dlk/muk gene expression in regenerating mouse liver

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 249, 927–932 (1998)ARTICLE NO. RC989249

Zonal Induction of Mixed Lineage Kinase ZPK/DLK/MUKGene Expression in Regenerating Mouse Liver

Melanie Douziech,* Gilles Grondin,* Anne Loranger,† Normand Marceau,† and Richard Blouin*,1

*Departement de Biologie, Faculte des Sciences, Universite de Sherbrooke, Sherbrooke, Quebec J1K 2R1, Canada; and†Centre de Recherche en Cancerologie de l’Universite Laval, l’Hotel-Dieu de Quebec, Quebec G1R 2J6, Canada

Received July 28, 1998

amino acid sequence identity in their kinase domain andZPK/DLK/MUK is a serine/threonine kinase believed similar overall structure. Typically, MLKs contain, in

to be involved in the regulation of cell growth and dif- addition to their catalytic domains, an amino-terminalferentiation. To further explore the suggested partici- src homology 3 (SH3) domain, two central leucine/isoleu-pation of ZPK/DLK/MUK in this process, we examined cine zipper motifs and a carboxy-terminal proline-richthe expression and cellular localization of ZPK/DLK/ region. Although little is known about the role of MLKs,MUK mRNA in regenerating mouse liver following par-

there is accumulating evidence indicating that ZPK/tial hepatectomy by ribonuclease protection assay andDLK/MUK, MLK3/SPRK/PTK1, MLK2/MST, and LZKin situ hybridization. The steady-state level of ZPK/are upstream regulators of the c-Jun N-terminal kinaseDLK/MUK mRNA was very low in normal and sham-(JNK)/stress-activated protein kinase (SAPK) subgroupoperated mouse livers, whereas a marked and tran-of mitogen-activated protein kinases (MAPKs) (10-17).sient increase was observed in the regenerating liver.

In line with the accumulating evidence indicating aWhile ZPK/DLK/MUK mRNAs were rarely detected inpivotal role of MLKs in cell growth regulation, our re-hepatocytes from all zones of the normal liver, hepato-cent work using in situ hybridization has demonstratedcytes of regenerating liver exhibit a gradient of expres-that the gene encoding ZPK/DLK/MUK exhibits re-sion ranging from low in the periportal zone, to inter-

mediate in the mid-zone, to high in the pericentral markable patterns of cell type- and developmentalzone. These findings demonstrate a transient stimula- stage-specific expression during mouse embryogenesistion of ZPK/DLK/MUK gene expression that correlates (18), the highest levels being present in neural struc-with the growth response of hepatocyte subpopula- tures and epithelial compartments of various organstions in regenerating liver. q 1998 Academic Press during late organogenesis. Moreover, in adult mouse

tissues, ZPK/DLK/MUK mRNAs were restricted to spe-cific populations of differentiated cells, includingamong others Purkinje cells of the cerebellum, chief

Serine/threonine kinases constitute a superfamily of cells of the gastric glands, enterocytes and Paneth cellsrelated enzymes that act as central components of kinase of the small intestine, acinar cells of the pancreas, andcascades involved in signaling pathways triggered by the hepatocytes of the liver (19).binding of growth factors, hormones, and cytokines to Because of its ability to regenerate, the liver hasdifferent cell-surface receptors. These kinases can be di- served as an important in vivo model for studying thevided into several subclasses based upon their structural mechanisms that control cell proliferation and differenti-similarities, substrate specificities and modes of regula- ation (20). Under normal physiological conditions, hepa-tion (1), and one such subclass is the recently discovered tocytes of adult liver have a long life span and rarelyfamily termed mixed lineage kinases (MLKs). Up to now, undergo cell division. However, when liver parenchymafive distinct MLK family members, named MLK1 (2), is lost by either surgical resection or chemical intoxica-MLK2/MST (3, 4), MLK3/SPRK/PTK1 (5–7), ZPK/DLK/ tion, the remaining hepatocytes rapidly reenter the cellMUK (8–10), and LZK (11) have been discovered and cycle and divide in an effort to restore the original masscharacterized biochemically. All members share 46-77% of hepatic tissue. In rats and mice, ú95% of hepatocytes

replicate after a 2/3 hepatectomy (PH), and the hepaticmass is entirely reconstituted within 7-10 days. To ex-1 To whom correspondence should be addressed at Departement plore the possibility that ZPK/DLK/MUK may be part ofde Biologie, Faculte des Sciences, Universite de Sherbrooke, 2500,the molecular mechanisms regulating cell proliferation,boulevard de l’Universite, Sherbrooke, Quebec, Canada J1K 2R1.

Fax: (819) 821-8049. E-mail: [email protected]. we have analyzed the expression of the ZPK/DLK/MUK

0006-291X/98 $25.00Copyright q 1998 by Academic PressAll rights of reproduction in any form reserved.

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Vol. 249, No. 3, 1998 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

gene in mouse liver following PH using both ribonucleaseprotection assay and in situ hybridization. The resultsdemonstrate a transient increase in ZPK/DLK/MUKmRNA expression in growth-stimulated hepatocytes, re-stricted to well defined zones of the liver lobules.

MATERIALS AND METHODS

Animals. FVB/N mice were used for all experiments. Some micewere subjected to PH (21) under anesthesia via a sodium pentobarbi-tal (54 mg/kg of body weight) intraperitoneal injection. Through amedian-line incision posteriorly from the xiphoid process of the ster-num, the large median lobe with the lateral lobe were ligated andthen excised, as described before (22). At various times post-opera-tion, the animals were sacrificed under anesthesia and the remainingliver tissue was sampled. A portion of the liver was processed for insitu hybridization, and the rest was stored in liquid nitrogen untilRNA isolation. For sham operations, mice were anesthetized, sub-jected to midventral laparotomy, and the liver was gently rubbedbetween the fingers but no tissue was excised (22). FIG. 1. RNAse protection analysis of ZPK/DLK/MUK gene expres-

sion in regenerating liver. A [32P]-labeled ZPK/DLK/MUK antisenseRibonuclease protection assay. Total RNA was isolated from liv-riboprobe was hybridized to 10 mg of total RNA isolated from control,ers using the acid phenol extraction method (23). An antisense ZPK/sham, and partially hepatectomized mouse liver 1, 2, 3, and 7 days afterDLK/MUK RNA probe riboprobe was generated from linearizedsurgery. To verify for RNA equivalence, a g-actin probe was included inpBluescript plasmid containing a 192-bp fragment of the mouse ZPK/the samples. Controls include ZPK/DLK/MUK probe hybridized withDLK/MUK cDNA (19) using T3 RNA polymerase and [a-32P]UTP.yeast RNA alone, treated (/) or not treated (0) with RNase.Hybridization and RNase digestion were performed using the RPA

II kit (Ambion Inc., Austin, Texas) in accordance with the conditionsrecommended by the manufacturer. Protected products were re-solved by electrophoresis in 5% acrylamide/7M urea gels. To control phosphate as substrate (Boehringer Mannheim, Canada). Photo-for RNA equivalence, a g-actin probe was included in the samples. graphs were taken under bright field illumination using a Zeiss pho-

tomicroscope and Kodak TMAX 100 print film.In situ RNA hybridization. Livers were fixed overnight in 4%paraformaldehyde, dehydrated with graded ethanol solutions, andembedded in paraffin. Paraffin sections were cut serially at 4 mm RESULTS AND DISCUSSIONthickness, mounted on polyionic slides (Superfrost Plus, Fisher, Can-ada) and kept at 47C. Sections were deparaffinized with xylene and

Total RNAs were extracted from the livers of normal,the tissues were rehydrated through graded ethanol solutions tosham-operated, and partially hepactomized adult micePBS. Sections were treated in 0.2 M HCl at room temperature for

10 min and then washed in PBS before immersion in 0.3% Triton X- and the levels of ZPK/DLK/MUK mRNA and g-actin100 in PBS for 15 minutes. After rinsing with the same buffer, the mRNA, used here as control, were evaluated by ribo-sections were incubated for 1 min in ice cold 20% acetic acid to reduce nuclease protection assay (Fig. 1). It should be stressednonspecific background and subsequently rinsed twice in PBS for 2

that the 32P-labeled ZPK/DLK/MUK and g-actin ribo-minutes. The slides were then digested with proteinase K (2 mg/ml)for 30 minutes at 377C. Digestion was stopped by washing in 0.2% probes were included in the same reaction to measureglycine in PBS, followed by a second fixation with paraformaldehyde relative levels of RNA present in each hybridization.(4%). The sections were rinsed in PBS, acetylated with triethanol- ZPK/DLK/MUK mRNAs were present at low levels inamine and washed again in PBS. Sections were prehybridized in 51 normal adult mouse liver. In contrast, a prominent in-SSC, 51 Denhardt’s solution, 50% formamide, 250 mg/ml yeast t-

crease in ZPK/DLK/MUK mRNAs was observed in theRNA, 250 mg/ml denatured salmon sperm DNA, and 4 mM EDTA at427C for 3 h. Tissues were then hybridized overnight at 427C in the regenerating liver, at 1 day after PH. It reached a peaksame solution (but without salmon sperm DNA) with either anti- on day-2 and then decreased progressively to controlsense or sense control mouse ZPK RNA probes at a concentration of levels at day-7. No significant change in ZPK/DLK/10 ng/ml. The riboprobes were generated from linearized pBluescript

MUK mRNA content was observed in sham-operatedKS/ plasmid (Stratagene, USA) containing a 192 bp fragment ofanimals. In the case of g-actin mRNAs, the level variedthe murine ZPK cDNA (19) using T3 (antisense) or T7 (sense) RNA

polymerase and digoxigenin-11-UTP (Boehringer Mannheim, Can- in the opposite way, i.e., high at day 0, low at day 2 andada). Following hybridization, the slides were washed twice in 21 back to normal level at day 7. This transient increase inSSC for 15 minutes at room temperature, incubated once for 10 min ZPK/DLK/MUK mRNAs correlates with the wave ofwith RNase A (1 mg/ml) at room temperature, rinsed twice in 21

DNA synthesis occuring in regenerating mouse liverSSC at room temperature for 15 min and finally washed twice in 11after PH (24), arguing for a particular role in growthSSC at 377C for 15 minutes. The sections were blocked for 60 min at

room temperature in 0.5% blocking reagent (Boehringer Mannheim, regulation. This view is supported by recent data show-Canada), followed by a 2 h incubation with alkaline phosphatase ing an increase in ZPK/DLK/MUK mRNAs during heal-conjugated anti-digoxigenin antibody diluted 1:500 in 100 mM Tris- ing in the rat femur fracture model (25).HCl, 150 mM NaCl (pH 7.5). After washing, the hybridized cells were

ZPK/DLK/MUK mRNA distribution patterns in nor-visualised by a standard immunoalkaline phosphatase reaction, us-ing nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl- mal and PH mouse liver sections at day-1 and day-2

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FIG. 2. Localization of ZPK/DLK/MUK mRNA in mouse liver tissue following partial hepatectomy. (A) In situ hybridization of ZPK/DLK/MUK antisense probe on liver sections of normal mice. Expression of ZPK/DLK/MUK mRNA is detected at very low levels throughoutthe entire hepatic parenchyma, with occasional hepatocytes being more intensely labeled (arrow). (B) In situ hybridization of ZPK/DLK/MUK antisense probe on liver sections 1 day after hepatectomy. A remarkable increase of ZPK/DLK/MUK gene expression is observed inhepatocytes surrounding the centrolobular vein (C) while much lower levels are detected in the portal area (P). (C) Hematoxylin-eosinstaining of a liver tissue section adjacent to the one shown in B. Original magnification: A, 1200; B and C, 1125. Bar Å 50 mm.

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FIG. 3. Localization of ZPK/DLK/MUK mRNA in mouse liver tissue following partial hepatectomy. (A) Hematoxylin-eosin staining ofa liver section 1 day after hepatectomy. The hematopoietic tissue is pointed by an arrow whereas portal area and the centrolobular veinare represented by a P and a C, respectively. (B) In situ hybridization of ZPK/DLK/MUK antisense probe on a liver section adjacent to theone shown in A. The expression of ZPK/DLK/MUK mRNA is detectable in hepatocytes surrounding both the central vein (C) and thehematopoietic tissue (arrow). Note the absence of signal in the hematopoietic tissue. Original magnification: 1200. Bar Å 50 mm.

post-surgery were determined by in situ hybridization The predominant localization of ZPK/DLK/MUKmRNAs in the pericentral zone of mouse liver paren-using single-strand digoxigenin-labeled ZPK/DLK/

MUK antisense and sense probes. Hybridization with chyma is of particular interest because it shows astrong correlation with zonal variations that exist inthe antisense riboprobe demonstrated a very low label-

ing throughout the entire parenchyma in normal liver the proliferative activity of hepatocytes from differentlobular localizations after partial hepatectomy (26). In(Fig. 2A). In comparison, the labeling was much

stronger after PH, with the signal at day-1 being prefer- fact, previous studies of kinetics of DNA synthesis andmitosis have indicated that pericentral hepatocytes areentially restricted to hepatocytes located around the

centrilobular veins and the mid-zone (Fig. 2B). Further delayed in their entry into S-phase after hepatic resec-tion when compared to hepatocytes from other zonesexamination at high power magnification clearly

showed a prominent labeling in the centrilobular zone, (27, 28). While the molecular basis underlying this dif-ference in cell cycle activation is unclear, there are dataan intermediate labeling in the mid-zone and a low

labeling around the portal zone (Fig. 4A). Moreover, it indicating that phenotypic variability of hepatocytesub-populations and zonal heterogeneity in the hepaticis worth noticing that no signal was observed over foci

of erythropoiesis (Fig. 3B). The pattern of ZPK/DLK/ microenvironment are involved at least partially (29).The heterogeneity in ZPK/DLK/MUK mRNA distribu-MUK mRNA distribution expression obtained at day-

1 was maintained within each liver lobule at day-2 tion observed after PH may somehow be related to theinherent differences in hepatocyte growth activityafter PH (Fig. 4B). Control hybridization with the sense

probe at day-2 after PH revealed no signal in any of along the porto-central axis of the hepatic lobules.Since ZPK/DLK/MUK is an activator of JNK/SAPKthese zones (Fig. 4C).

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FIG. 4. Localization of ZPK/DLK/MUK mRNA in mouse liver tissue section 2 days after partial hepatectomy. (A) In situ hybridizationof ZPK/DLK/MUK antisense probe on a liver tissue section 1 day after hepatectomy. (B) In situ hybridization of ZPK/DLK/MUK antisenseprobe on a liver tissue section 2 days after hepatectomy. A high level of ZPK/DLK/MUK mRNA expression is detectable in the areasurrounding the central vein (C). (C) In situ hybridization of ZPK/DLK/MUK sense probe on liver sections 2 days after hepatectomy. Notethe absence of labeling in all hepatocytes. Original magnification: 1200. Bar Å 50 mm.

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6. Gallo, K. A., Mark, M. R., Scadden, D. T., Wang, Z., Gu, Q., and(10,12), its involvement in growth regulation is likelyGodowski, P. J. (1994) J. Biol. Chem. 269, 15092–15100.to take place via JNK/SAPK activation. Although the

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Oncogene 12, 641–650.activated not only by growth-promoting factors, but also11. Sakuma, H., Ikeda, A., Oka, S., Kozutsumi, Y., Zanetta, J. P.,by cytokines and various cellular stresses known to in-

and Kawasaki, T. (1997) J. Biol. Chem. 272, 28622–28629.duce growth arrest or apoptosis. In this regard, several 12. Fan, G., Merritt, S. E., Kortenjann, M., Shaw, P. E., and Holz-external activators of the JNK/SAPK pathway, such as man, L. B. (1996) J. Biol. Chem. 271, 24788–24793.epidermal growth factor and tumor necrosis factor-a, 13. Tibbles, L. A., Ing, Y. L., Kiefer, F., Chan, J., Iscove, N., Wood-stimulate hepatocyte growth, whereas other factors like gett, J. R., and Lassam, N. J. (1996) EMBO J. 15, 7026–7035.

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15. Rana, A., Gallo, K., Godowski, P., Hirai, S.-i., Ohno, S., Zon, L.,DLK/MUK may transduce inhibitory signals for cell pro-Kyriakis, J. M., and Avruch, J. (1996) J. Biol. Chem. 271, 19025–liferation during liver regeneration is supported by the 19028.

finding that its overexpression in fibroblasts causes 16. Hirai, S.-i., Katoh, M., Terada, M., Kyriakis, J. M., Zon, L. I.,growth arrest (30). Taken together the data suggest that Rana, A., Avruch, J., and Ohno, S. (1997) J. Biol. Chem. 272,

15167–15173.the transient induction of ZPK/DLK/MUK gene expres-17. Minden, A., and Karin, M. (1997) Biochim. Biophys. Acta 1333,sion in hepatocyte subpopulations is part of sequential

F85–F104.growth on/off signaling events taking place during liver18. Nadeau, A., Grondin, G., and Blouin, R. (1997) J. Histochem.regeneration.

Cytochem. 45, 107–118.19. Blouin, R., Beaudoin, J., Bergeron, P., Nadeau, A., and Grondin,

G. (1996) DNA Cell Biol. 15, 631–642.ACKNOWLEDGMENTS20. Michalopoulos, G. K., and DeFrances, M. C. (1997) Science 276,

60–66.The authors thank Mrs. Sheila MacLean for critical reading of the 21. Higgins, G. M., and Anderson, R. M. (1931) Arch. Pathol. 12,

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22. Loranger, A., Duclos, S., Grenier, A., Price, J., Wilson-Heiner,the Medical Research Council of Canada (N.M.). M.D. is a recipient ofM., Baribault, H., and Marceau, N. (1997) Am. J. Pathol. 151,a studentship from the Natural Sciences and Engineering Research1673–1683.Council of Canada.

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zonal induction of mixed lineage kinase zpk/dlk/muk gene expression in regenerating mouse liver

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