Developmental and growth-related regulation of expression of serine dehydratase mRNA in rat liver

In rat liver, serine dehydratase mRNA is undetectable in the late prenatal period, but its level increases rapidly after birth to a transient peak, and then after decrease gradually increases again to a maximum 2 weeks after birth that is slightly higher than that of adult liver. To determine whether mature quiescent hepatocytes proliferate without loss of differentiated functions, we measured the serine dehydratase mRNA contents in regenerating liver and primary cultured hepatocytes from adult rats. Partial hepatectomy resulted in a dramatic decrease in the mRNA content within 24 h and then its recovery within a week. In subconfluent cultures of adult rat hepatocytes that did not grow even in the presence of mitogens, serine dehydratase mRNA was maintained at a high level. However, when the hepatocytes were cultured at low cell density without added mitogens, their serine dehydratase mRNA content decreases to a quarter of that of subconfluent cultures. The possibility that the expression of serine dehydratase mRNA is regulated in G0/G1 transition before entry into the S phase and the relationship of the mRNA with growth are discussed.     

Transcriptional enhancement of UDP-glucuronosyltransferase form 1A2 (UGT1A2) by nuclear factor I-A (NFI-A) in rat hepatocytes

In cultured primary hepatocytes UDP-glucuronosyltransferase form 1A2 (UGT1A2) mRNA level is 80 times higher than that found in rat liver. We previously identified an enhancer sequence in the UGT1A2 promoter, and designated it as culture-associated expression responsive enhancer module (CEREM). Affinity chromatography with DNA fragments containing CEREM allowed enrichment of nuclear factor I (NFI) proteins from cultured hepatocytes. The NFI family is encoded by four distinct genes, NFI-A, NFI-B, NFI-C, and NFI-X. Immunoblot analysis with isoform-specific antibodies showed that NFI-A1 existed as a major component in rat liver and cultured hepatocytes. By contrast, NFI-C1 was present in rat liver but disappeared immediately upon cultivation of hepatocytes. Only trace amounts of NFI-B and NFI-X were detectable in rat liver and cultured hepatocytes. NFI-A1 elevated expression of the reporter gene that is under the control of CEREM, while NFI-C1 had an inhibitory effect. Co-expression of a constant amount of NFI-A1 with an increasing amount of NFI-C1 led to a concentration-dependent decrease in the expression of the CEREM-controlled reporter gene mediated by NFI-A1. Activation of UGT1A2 expression by NFI-A1 is suppressed by the coexistence of NFI-C1 in the liver, and culture-associated expression of UGT1A2 is triggered by the rapid disappearance of NFI-C1 in cultured hepatocytes.     

Annexin A3 expression increases in hepatocytes and is regulated by hepatocyte growth factor in rat liver regeneration

Annexin (Anx) A3 increases and plays important roles in the signalling cascade in hepatocyte growth in cultured hepatocytes. However, no information is available on its expression and role in rat liver regeneration. In the present study, AnxA3 expression was investigated to determine whether it also plays a role in the signalling cascade in rat liver regeneration. AnxA3 protein and mRNA level both increase in liver after administration of carbon tetrachloride (CCl4) or 70% partial hepatectomy. AnxA3 protein level increases in isolated parenchymal hepatocytes, but not in non-parenchymal liver cells, in these rat liver regeneration models. AnxA3 mRNA increases in hepatocytes after CCl4 administration. Anti-hepatocyte growth factor antibody suppresses this increase in AnxA3 mRNA level. These results demonstrate that AnxA3 expression increases in hepatocytes through a hepatocyte growth factor-mediated pathway in rat liver regeneration models, suggesting that AnxA3 plays an important role in the signalling cascade in rat liver regeneration.     

Differential regulation of glucose transporter 1 and 2 mRNA expression by epidermal growth factor and transforming growth factor-beta in rat hepatocytes.

We have examined by Northern blot analysis the expression of two members of the glucose transporter family of genes (GLUT-1 and GLUT-2) in regenerating liver and in hepatocytes cultured under various conditions. GLUT-1, although thought to be a growth-associated gene, is not expressed in normal or regenerating liver, whereas GLUT-2, a liver-specific gene, is abundant in normal liver and gradually up-regulated during liver regeneration. Conversely, in hepatocytes cultured conventionally on dried rat tail collagen (RTC) in the presence of EGF and insulin, which potentiate proliferation, GLUT-1 mRNA is rapidly and abundantly expressed, whereas GLUT-2 is depressed. To investigate the causes of this "switch" in glucose transporter expression seen when hepatocytes are removed from the liver and cultured under the conventional proliferative conditions, we examined the effects of specific growth factors and extracellular matrices on cultured hepatocytes. EGF, a potent liver mitogen, although causing a threefold induction of GLUT-1, was found to have no effect on GLUT-2 expression, suggesting that the increase in GLUT-2 seen in regenerating liver is not due to EGF. Inhibition of protein synthesis by cycloheximide in cultured hepatocytes does not prevent the induction of GLUT-1 mRNA. In addition, treatment of cells with cycloheximide appears to stabilize the GLUT-2 mRNA, preventing the usual down-regulation of this gene in cultured hepatocytes. The expression of the two glucose transporter mRNAs also differed when the hepatocytes were adherent to particular cell matrices. Culture of hepatocytes on a reconstituted basement membrane gel matrix (EHS) is known to restrain their growth and mediate high levels of differentiated hepatocytic functions that are lost under conventional culture conditions. Unlike cells on RTC, hepatocytes on EHS expressed low levels of GLUT-1 mRNA, and decreased GLUT-2 mRNA. TGF-beta, an attenuator of DNA synthesis, when added to cultures on RTC, substantially down-regulated GLUT-2 but had no effect on GLUT-1. We propose that the effectors, EGF, TGF-beta and basement membrane components, play a significant role in the regulation of expression of GLUT-1 and GLUT-2 in hepatocytes.     

Spatiotemporal changes in Ha-ras p21 expression through the hepatocyte cell cycle during liver regeneration.

The protein product of the ras oncogene, Ha-ras (p21), is thought to be an important regulator of cell growth. The cytoplasmic relocalization of p21 in the cell during the cell cycle suggests a transient signaling role for this protein in association with its signal transduction function. Because of the importance of this role we examined spatial patterns in vivo of p21 expression at the protein and mRNA levels in hepatocytes during compensatory growth in rat liver following partial hepatectomy. A low level of p21 was immunolocalized on the cytoplasmic membrane of nonregenerating hepatocytes. The level of hepatic p21 increased significantly and without spatial restriction within the liver from 36 to 60 hr after partial hepatectomy (PH). p21 was localized in the cytoplasm of dividing hepatocytes and on the hepatic cytoplasmic membrane. The elevated p21 level decreased and was found mainly on hepatocyte plasma membranes by 96 hr after PH. Immunogold electron microscopy showed p21 localized over mitochondrial membranes and nuclei in nondividing regenerating hepatocytes. Approximately 50% of nonregenerating hepatocytes show nuclear localization of p21. This percentage changes with time following PH. The decrease in nuclear localization was accompanied with an increase in the low number of hepatocytes which demonstrated cytoplasmic localization in nondividing hepatocytes in regenerating liver. Flow cytometric analysis revealed a significant increase of p21 at 36 hr after PH which was 12 hr after the initial induction of ras mRNA. ras mRNA level increased 1.5-fold at 24 hr after PH and a maximum twofold induction was observed at 48 hr. Cell-cycle analysis of regenerating hepatocytes indicated a synchronized first peak of cell division 36-40 hr after PH. Dual parameter flow cytometry revealed that the level of p21 in hepatocytes in S phase and G2/M phase of the cell cycle was significantly higher than that in G0/G1 phase during regeneration. These findings suggest that p21 is important for the progression of regenerating hepatocytes to S phase and then to G2/M phase.     

IL-6 functions as an exocrine hormone in inflammation. Hepatocytes undergoing acute phase responses require exogenous IL-6

We have previously shown that IL-6 is the major monocyte- and fibroblast-derived regulator of acute phase protein gene expression and synthesis in hepatocytes in inflammation. Recently, we and others have shown that rat and human hepatoma cells express IL-6 mRNA, and the question arose as to whether normal hepatocytes express IL-6 and whether any such expression occurs under normal physiologic conditions or is seen in inflammation. Poly A+ mRNA of liver from normal rats and from rats undergoing an acute phase response was not positive when probed with cDNA for rat IL-6 under conditions in which macrophage mRNA was strongly positive. We then compared poly A+ mRNA from purified hepatocytes freshly isolated from normal rats--from rats that were undergoing an acute inflammatory response and from freshly isolated normal hepatocytes that had been cultured for 24 h in the presence or absence of dexamethasone (microM). Only the mRNA from normal hepatocytes cultured for 24 h in the absence of any glucocorticoid was obviously positive for IL-6. The increased expression of gamma-fibrinogen mRNA indicated the presence of inflammation. These results confirm the identification of IL-6 as an exogenous hormone for regulating normal hepatic acute phase protein synthesis in inflammation and rules out an autocrine mechanism being active in the liver in normal homeostasis.     

TEC酪氨酸激酶基因是一种与肝再生调控相关的早期反应基因

 肝再生过程中立即早期反应基因的表达在成熟肝细胞由G0 期向G1期的转变中起着关键作用 .为探讨肝再生早期基因表达的变化 ,利用表达性差异显示分析 (RDA)技术研究了 2 3肝部分切除后 1h再生肝选择性基因表达 ,发现一株TEC酪氨酸激酶同源序列存在于差减产物中 ,RNA狭缝杂交证实确为差异表达基因 .从大鼠肝cDNA文库中分离其全长cDNA ,序列分析结果表明 ,该基因为小鼠 人TEC酪氨酸激酶的同源体 ,进而以该cDNA为探针 ,用Northern杂交证实 2 3肝部分切除后TEC酪氨酸激酶基因在 1h内呈现瞬间表达增加 ,其表达水平较基础水平增高 2 5倍 ;在原代培养大鼠肝细胞体系中 ,EGF可迅速诱导TEC基因表达 ,且不被蛋白合成抑制剂阻断 .结果表明 ,TEC基因是一种与肝再生调控密切相关的早期反应基因 .     

Effect of Collagen Gel Configuration on the Cytoskeleton in Cultured Rat Hepatocytes

The cytoskeleton is important in the maintenance of cellular morphology and differentiated function in a number of cell types, including hepatocytes. In this study, adult rat hepatocytes sandwiched between two layers of collagen gel were compared to cells cultured on a single collagen gel for differences in the organization and expression of the cytoskeletal proteins actin and tubulin. Hepatocytes cultured between two layers of hydrated rat tail tendon collagen (sandwich gel) morphologically resembled cells in intact liver for several weeks. Actin filaments (F-actin) in these hepatocytes were concentrated under the plasma membrane in regions of cell-cell contact. In contrast, hepatocytes cultured on a single collagen gel were flattened and motile and had F-actin containing stress fibers. This was accompanied by a severalfold increase in actin mRNA. Microtubules formed an interwoven network in hepatocytes cultured in a sandwich gel, but in single gel cultures they formed long parallel arrays extending out to the cell periphery. Tubulin mRNA was severalfold greater in hepatocytes cultured on a single gel. Fibronectin and laminin staining were greater in single gel cultures, and these proteins were concentrated in fibrils radiating from the cell periphery. Overlaying a second collagen gel onto hepatocytes that had been cultured on a single gel (double gel rescue) reversed cell spreading and reduced stress fibers. Double gel rescue also resulted in a decrease in actin and tubulin mRNA to levels present in sandwich gel cultures and freshly isolated hepatocytes. These results show that the configuration of the external matrix has a dynamic effect on cytoskeletal proteins in cultured rat hepatocytes.     

Spatiotemporal changes in Ha-ras p21 expression through the hepatocyte cell cycle during liver regeneration

The protein product of the ras oncogene, Ha-ras (p21), is thought to be an important regulator of cell growth. The cytoplasmic relocalization of p21 in the cell during the cell cycle suggests a transient signaling role for this protein in association with its signal transduction function. Because of the importance of this role we examined spatial patterns in vivo of p21 expression at the protein and mRNA levels in hepatocytes during compensatory growth in rat liver following partial hepatectomy. A low level of p21 was immunolocalized on the cytoplasmic membrane of nonregenerating hepatocytes. The level of hepatic p21 increased significantly and without spatial restriction within the liver from 36 to 60 hr after partial hepatectomy (PH). p21 was localized in the cytoplasm of dividing hepatocytes and on the hepatic cytoplasmic membrane. The elevated p21 level decreased and was found mainly on hepatocyte plasma membranes by 96 hr after PH. Immunogold electron microscopy showed p21 localized over mitochondrial membranes and nuclei in nondividing regenerating hepatocytes. Approximately 50% of nonregenerating hepatocytes show nuclear localization of p21. This percentage changes with time following PH. The decrease in nuclear localization was accompanied with an increase in the low number of hepatocytes which demonstrated cytoplasmic localization in nondividing hepatocytes in regenerating liver. Flow cytometric analysis revealed a significant increase of p21 at 36 hr after PH which was 12 hr after the initial induction of ras mRNA. ras mRNA level increased 1.5-fold at 24 hr after PH and a maximum twofold induction was observed at 48 hr. Cell-cycle analysis of regenerating hepatocytes indicated a synchronized first peak of cell division 36–40 hr after PH. Dual parameter flow cytometry revealed that the level of p21 in hepatocytes in S phase and G₂/M phase of the cell cycle was significantly higher than that in G₀/G₁ phase during regeneration. These findings suggest that p21 is important for the progression of regenerating hepatocytes to S phase and then to G₂/M phase.     

Expressions of the c-Ha-ras and c-myc genes in rat liver tumors

Expressions of the c-Ha-ras and c-myc genes were studied by Northern blotting of total RNA from primary tumors and non-tumorous parts of the liver of rats given diet containing 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB) and from established rat hepatoma cell lines. The expression of the c-Ha-ras gene was found to be high in the primary tumors, non-tumorous parts of 3'-Me-DAB-treated livers and hepatoma cell lines. In contrast, the c-myc gene was expressed at a high level only in primary tumors and hepatoma cell lines. During 3'-Me-DAB treatment, the c-Ha-ras mRNA level in the liver increased by day 5 and then remained high. Increase in expression of the c-Ha-ras gene in regenerating liver was confirmed. These findings suggest that increase in expression of the c-Ha-ras gene is related to proliferation of hepatocytes, whereas expression of the c-myc gene is associated with hepatocarcinogenesis.     

Expression of c-fos oncogene during hepatocarcinogenesis, liver regeneration and in synchronized HTC cells

We have studied the expression of c-fos gene in rat hepatoma induced by DENA. An increase of c-fos mRNA concentration was observed after 8 days, but the maximal 5- to 6-fold increase was observed after 70 weeks. This increase was found in perinodular hepatocytes as well as in cancer nodules. c-fos expression was also enhanced during liver regeneration at a period corresponding to cell proliferation. In HTC cells the arrest of the cell cycle at early G1 phase by addition of sodium butyrate was accompanied by a strong increase of c-fos gene expression. However the c-fos mRNA rapidly decreased after removal of sodium butyrate during the progression of the cells in the cell cycle and increased transiently when the cells entered again in G1 phase.     

High Responsiveness to Thyroid Hormone of Adult Rat Primary Hepatocytes Cultured on EHS-Gel

The induction of malic enzyme gene expression by triiodothyronine and insulin was severely blunted in rat monolayer hepatocytes cultured on type I collagen compared with that in spherical hepatocytes cultured on a reconstituted basement membrane gel (EHS-gel). Although the mRNA level of thyroid hormone receptor β (TRβ) gradually decreased in the monolayer hepatocytes during culture, the mRNA level in the hepatocytes on EHS-gel was maintained at around the in vivo level. Our results suggest that the maintenance of TRβ mRNA on EHS-gel is responsible for the high responsiveness to thyroid hormone in a hepatocyte culture.     

Induction of sodium pump beta 1-subunit mRNA expression during hepatocellular growth transitions in vitro and in vivo.

Previous suggestions (Hubert, J. J., Schenk, D. B., Skelly, H., and Leffert, H. L. (1986) Biochemistry 25, 4156-4163) of tissue-specific isoforms or nonexistence of hepatic Na,K-ATPase beta 1-subunits were reevaluated by quantifying beta 1-subunit mRNA levels in quiescent and proliferating liver. RNA was extracted from caudate liver lobes of sham or 67% hepatectomized adult rats and from primary cultures of adult rat hepatocytes that simulate developmental and regenerating growth transitions. Northern blot analysis with a 32P-labeled full-length Na,K-ATPase beta 1-cDNA probe (Mercer, R. W., Schneider, J. W., Savitz, A., Emmanuel, J., Benz, T.J., and Levenson, R. (1986) Mol. Cell. Biol. 6, 3884-3890) revealed four (approximately 2.7, 2.4, 1.7-1.8, and 1.5 kilobases) low abundance mRNA species in quiescent tissue, freshly isolated hepatocytes, and cultured hepatocytes derived from lag or late stationary phase (1-2 days or 11-12 days postplating, respectively). In contrast, proliferating liver from 4 h post-67% hepatectomized rats or cultured hepatocytes in logarithmic growth phase contained levels of beta 1-subunit mRNA which exceeded quiescent levels by 4-35-fold. Membrane Na,K-ATPase activity also increased 2-3-fold during liver regeneration 12-24 h after partial hepatectomy. When proliferation in vitro was augmented by transforming growth factor-alpha, a hepatocyte mitogen, or reinitiated in late stationary phase by a change to fresh culture medium containing rat serum, beta 1-subunit mRNA expression was restimulated 4-20-fold. Parallel measurements of alpha-tubulin mRNA induction showed relatively nonsynchronous or invariant changes during hepatocyte proliferative transitions; similar results were obtained after Northern blots with a sodium pump alpha I-subunit cDNA probe. No detectable hybridization signals were observed when either rat kidney or hepatocyte RNAs from freshly isolated and cultured cells or regenerating tissues were probed for the sodium pump 3.4-kilobase mRNA beta 2-isoform. These observations suggest that enhanced hepatic beta 1-subunit gene expression is linked specifically to growth-associated increases in Na,K-ATPase activity, hepatocyte proliferation, and mitogen activation.