Pleckstrin Homology (PH) Domain Leucine-rich Repeat Protein Phosphatase Controls Cell Polarity by Negatively Regulating the Activity of Atypical Protein Kinase C.

The proper establishment of epithelial polarity allows cells to sense and respond to signals that arise from the microenvironment in a spatiotemporally controlled manner. Atypical PKCs (aPKCs) are implicated as key regulators of epithelial polarity. However, the molecular mechanism underlying the negative regulation of aPKCs remains largely unknown. In this study, we demonstrated that PH domain leucine-rich repeat protein phosphatase (PHLPP), a novel family of Ser/Thr protein phosphatases, plays an important role in regulating epithelial polarity by controlling the phosphorylation of both aPKC isoforms. Altered expression of PHLPP1 or PHLPP2 disrupted polarization of Caco2 cells grown in 3D cell cultures as indicated by the formation of aberrant multi-lumen structures. Overexpression of PHLPP resulted in a decrease in aPKC phosphorylation at both the activation loop and the turn motif sites; conversely, knockdown of PHLPP increased aPKC phosphorylation. Moreover, in vitro dephosphorylation experiments revealed that both aPKC isoforms were substrates of PHLPP. Interestingly, knockdown of PKCζ, but not PKCι, led to similar disruption of the polarized lumen structure, suggesting that PKCζ likely controls the polarization process of Caco2 cells. Furthermore, knockdown of PHLPP altered the apical membrane localization of aPKCs and reduced the formation of aPKC-Par3 complex. Taken together, our results identify a novel role of PHLPP in regulating aPKC and cell polarity.

Loss of PHLPP protects against colitis by inhibiting intestinal epithelial cell apoptosis.

A common feature of inflammatory bowel disease (IBD) is the loss of intestinal epithelial barrier function due to excessive apoptosis of intestinal epithelial cells (IECs). However, the molecular mechanism underlying increased IEC apoptosis remains unclear. Here, we investigated the role of PHLPP, a novel family of protein phosphatases, in regulating inflammation-induced IEC apoptosis in mouse models of colitis. Both Phlpp1 and Phlpp2 genes were deleted in mice. Compared with wild-type mice, PHLPP double knockout (DKO) mice were protected from colitis induced by DSS as demonstrated by lower histopathological scores, and this reduced susceptibility to colitis was associated with decreased apoptosis and increased Akt activity in IECs in vivo. In addition, epithelial organoids derived from PHLPP DKO mice were more resistant to inflammation-induced apoptosis while inhibition of Akt activity abolished the protective effect of PHLPP-loss. Furthermore, we found that PHLPP expression was significantly reduced in IECs following the induction of colitis by DSS and in human IBD patient samples. This inflammation-induced downregulation of PHLPP was partially blocked by treating cells with a proteasome inhibitor. Taken together, our results indicated that proteasome-mediated degradation of PHLPP at the onset of inflammation plays an important role in protecting IEC injury by inhibiting apoptosis.

Hepatic expression profile of forkhead transcription factor genes in normal Balb/c mice and their dynamic changes after bile duct ligation.

Dysregulation of Forkhead box (Fox) transcription factor family genes was previously shown to lead to congenital disorders, diabetes mellitus, and carcinogenesis, and recent reports suggested that several Fox genes play important roles in the pathogenesis of liver fibrosis. The present study was initiated to determine the expression profiles of the Fox genes in normal Balb/c mouse liver and their dynamic expression changes during fibrogenesis induced by experimental bile duct ligation (BDL). RT-PCR was employed to detect 18 FOX family members including FOXO1, FOXO3, FOXM1, and FOXL1 in normal mouse liver. FQ-PCR was performed to analyze the dynamic mRNA expression changes of nine inflammation- or proliferation-related FOX family genes in BDL mice. Results showed that all the 18 Fox genes were expressed in the normal mouse liver, among which the expression of FOXO1 and FOXO3 were found to be the highest. The inflammation and proliferation-related FOX family genes were found to be dynamically changed during BDL-induced liver injury with reduced FOXO1 and enhanced FOXOL1 and FOXM1, indicating their potential involvement in the pathogenesis of liver fibrosis. This is the first systematic evaluation of hepatic expression of FOX genes in both normal and BDL mice.

Downregulation of PHLPP induced by endoplasmic reticulum stress promotes eIF2α phosphorylation and chemoresistance in colon cancer.

Aberrant activation of endoplasmic reticulum (ER) stress by extrinsic and intrinsic factors contributes to tumorigenesis and resistance to chemotherapies in various cancer types. Our previous studies have shown that the downregulation of PHLPP, a novel family of Ser/Thr protein phosphatases, promotes tumor initiation, and progression. Here we investigated the functional interaction between the ER stress and PHLPP expression in colon cancer. We found that induction of ER stress significantly decreased the expression of PHLPP proteins through a proteasome-dependent mechanism. Knockdown of PHLPP increased the phosphorylation of eIF2α as well as the expression of autophagy-associated genes downstream of the eIF2α/ATF4 signaling pathway. In addition, results from immunoprecipitation experiments showed that PHLPP interacted with eIF2α and this interaction was enhanced by ER stress. Functionally, knockdown of PHLPP improved cell survival under ER stress conditions, whereas overexpression of a degradation-resistant mutant PHLPP1 had the opposite effect. Taken together, our studies identified ER stress as a novel mechanism that triggers PHLPP downregulation; and PHLPP-loss promotes chemoresistance by upregulating the eIF2α/ATF4 signaling axis in colon cancer cells.

Upregulation of CPT1A is essential for the tumor-promoting effect of adipocytes in colon cancer.

Colon tumors grow in an adipose tissue-enriched microenvironment. Locally advanced colon cancers often invade into surrounding adipose tissue with a direct contact with adipocytes. We have previously shown that adipocytes promote tumor growth by modulating cellular metabolism. Here we demonstrate that carnitine palmitoyltransferase I (CPT1A), a key enzyme controlling fatty acid oxidation (FAO), was upregulated in colon cancer cells upon exposure to adipocytes or fatty acids. In addition, CPT1A expression was increased in invasive tumor cells within the adipose tissue compared to tumors without direct contact with adipocytes. Silencing CPT1A abolished the protective effect provided by fatty acids against nutrient deprivation and reduced tumor organoid formation in 3D culture and the expression of genes associated with cancer stem cells downstream of Wnt/ß-catenin. Mechanistically, CPT1A-dependent FAO promoted the acetylation and nuclear translocation of ß-catenin. Furthermore, knockdown of CPT1A blocked the tumor-promoting effect of adipocytes in vivo and inhibited xenograft tumor initiation. Taken together, our findings identify CPT1A-depedent FAO as an essential metabolic pathway that enables the interaction between adipocytes and colon cancer cells.

Inhibition of protein tyrosine phosphatase receptor type F suppresses Wnt signaling in colorectal cancer.

Wnt signaling dysregulation promotes tumorigenesis in colorectal cancer (CRC). We investigated the role of PTPRF, a receptor-type tyrosine phosphatase, in regulating Wnt signaling in CRC. Knockdown of PTPRF decreased cell proliferation in patient-derived primary colon cancer cells and established CRC cell lines. In addition, the rate of proliferation as well as colony formation ability were significantly decreased in tumor organoids grown in 3D, whereas the number of differentiated tumor organoids were markedly increased. Consistently, knockdown of PTPRF resulted in a decrease in the expression of genes associated with cancer stem cells downstream of Wnt/ß-catenin signaling. Treating PTPRF knockdown cells with GSK3 inhibitor rescued the expression of Wnt target genes suggesting that PTPRF functions upstream of the ß-catenin destruction complex. PTPRF was found to interact with LRP6 and silencing PTPRF largely decreased the activation of LRP6. Interestingly, this PTPRF-mediated activation of Wnt signaling was blocked in cells treated with clathrin endocytosis inhibitor. Furthermore, knockdown of PTPRF inhibited xenograft tumor growth in vivo and decreased the expression of Wnt target genes. Taken together, our studies identify a novel role of PTPRF as an oncogenic protein phosphatase in supporting the activation of Wnt signaling in CRC.

Enhanced glucose synthesis in three-dimensional hepatocyte collagen matrix.

Three-dimensional (3D) cell culture model offers a unique opportunity to study hepatocytes that require extracellular matrix to keep the cells at the differentiated state. In this report, we cultured isolated mouse hepatocytes in a 3D collagen matrix system and developed a protocol to measure glucose production at 3h, 6h, 18h and 24h after culture. The results demonstrated that hepatocytes cultured under 3D collagen matrix condition consistently produced glucose at 240-290 mg/10(6) cells for up to 24h. Contrarily, hepatocytes cultured under traditional monolayer condition produced less than 50 mg/10(6) cells glucose. We demonstrated higher expression of phosphoenolpyruvate carboxykinase (PEPCK), a key enzyme for the synthesis of glucose from pyruvate, and CCAAT/enhancer-binding protein alpha (C/EBPalpha), an important liver-specific transcription factor, under the 3D collagen matrix culture condition in comparison to the monolayer condition. Thus, the 3D collagen matrix system preserved metabolic function of hepatocytes and can be used as an in vitro model for studying hepatocyte glucose production and gluconeogenesis.

The mitochondrial retrograde signaling regulates Wnt signaling to promote tumorigenesis in colon cancer.

Cancer cells are known to upregulate aerobic glycolysis to promote growth, proliferation, and survival. However, the role of mitochondrial respiration in tumorigenesis remains elusive. Here we report that inhibition of mitochondrial function by silencing TFAM, a key transcription factor essential for mitochondrial DNA (mtDNA) replication and the transcription of mtDNA-encoded genes, markedly reduced tumor-initiating potential of colon cancer cells. Knockdown of TFAM significantly decreased mitochondrial respiration in colon cancer cells; however, the cellular levels of ATP remained largely unchanged as a result of increased glycolysis. This metabolic alteration rendered cancer cells highly susceptible to glucose deprivation. Interestingly, upregulation of glycolysis was independent of hypoxia-inducible factor-1 (HIF1) as TFAM knockdown cells fail to stabilize HIF1α under hypoxic conditions. Moreover, knockdown of TFAM results in decreased expression of genes-associated cancer stem cells downstream of Wnt/ß-catenin signaling. Metabolic analysis reveals that the level of α-ketoglutarate (α-KG) was significantly upregulated in TFAM knockout cells. Silencing of prolyl hydroxylase domain-containing protein 2 (PHD2), a α-KG-dependent dioxyenase, rescued the expression of target genes of both HIF1α and Wnt/ß-catenin. Furthermore, intestinal-specific knockout of TFAM prevents tumor formation in Apc-mutant mouse models of colon cancer. Taken together, our findings identify a novel role of mitochondria-mediated retrograde signaling in regulating Wnt signaling and tumor initiation in colon cancer.

[The clinical significance of real-time fluorescence PCR quantification of hepatocyte growth factor mRNA expression in acute leukemia].

OBJECTIVE: To detect quantitatively hepatocyte growth factor (HGF) mRNA expressions of bone marrow mononuclear cells (MNCs) in acute leukemia (AL) and investigate its clinical significance. METHODS: Total mRNA of quantitated bone marrow MNCs isolated from 67 de novo AL cases was extracted and then cDNA was synthesized. Expression of HGF mRNA was quantified absolutely using real-time fluorescence quantification PCR (FQ-PCR). RESULTS: Expressions of HGF mRNA in a group of AL were higher significantly than these in a control group (6.936 +/- 1.613, 0.407 +/- 0.170, P < 0.001), but there was similarity between a group of acute myeloid leukemia (AML) and group of acute lymphoblastic leukemia (ALL) (7.127 +/- 1.911, 6.635 +/- 0.934, P > 0.05). In AL subtypes, the expression of M5 (9.998 +/- 1.454) was higher than that of M2, M3, M4, L1, L2 and L3 (P < 0.001), but there were no differences among the latters (P > 0.05). Meanwhile, there was no statistical significance on the expressions of HGF mRNA between different age and sex (P > 0.05). In addition, expressions of HGF mRNA in the remission group were lower than these in the non-remission group (6.393 +/- 1.165, 8.041 +/- 1.848, P < 0.005). CONCLUSIONS: There are statistical significances of the expressions of bone marrow MNCs HGF mRNA among the AL group and control group. As to AL subtypes, there are no statistically significant differences between AML and ALL as well as between different age and sex. Besides, lower HGF mRNA level is correlated with better curative effect. It is suggested that HGF mRNA is a suitable index for AL diagnosis and treatment.

Downregulation of SREBP inhibits tumor growth and initiation by altering cellular metabolism in colon cancer.

Sterol regulatory element-binding proteins (SREBPs) belong to a family of transcription factors that regulate the expression of genes required for the synthesis of fatty acids and cholesterol. Three SREBP isoforms, SREBP1a, SREBP1c, and SREBP2, have been identified in mammalian cells. SREBP1a and SREBP1c are derived from a single gene through the use of alternative transcription start sites. Here we investigated the role of SREBP-mediated lipogenesis in regulating tumor growth and initiation in colon cancer. Knockdown of either SREBP1 or SREBP2 decreased levels of fatty acids as a result of decreased expression of SREBP target genes required for lipid biosynthesis in colon cancer cells. Bioenergetic analysis revealed that silencing SREBP1 or SREBP2 expression reduced the mitochondrial respiration, glycolysis, as well as fatty acid oxidation indicating an alteration in cellular metabolism. Consequently, the rate of cell proliferation and the ability of cancer cells to form tumor spheroids in suspension culture were significantly decreased. Similar results were obtained in colon cancer cells in which the proteolytic activation of SREBP was blocked. Importantly, knockdown of either SREBP1 or SREBP2 inhibited xenograft tumor growth in vivo and decreased the expression of genes associated with cancer stem cells. Taken together, our findings establish the molecular basis of SREBP-dependent metabolic regulation and provide a rationale for targeting lipid biosynthesis as a promising approach in colon cancer treatment.

Erbin Suppresses KSR1-Mediated RAS/RAF Signaling and Tumorigenesis in Colorectal Cancer.

Erbin belongs to the LAP (leucine-rich repeat and PDZ domain) family of scaffolding proteins that plays important roles in orchestrating cell signaling. Here, we show that Erbin functions as a tumor suppressor in colorectal cancer. Analysis of Erbin expression in colorectal cancer patient specimens revealed that Erbin was downregulated at both mRNA and protein levels in tumor tissues. Knockdown of Erbin disrupted epithelial cell polarity and increased cell proliferation in 3D culture. In addition, silencing Erbin resulted in increased amplitude and duration of signaling through Akt and RAS/RAF pathways. Erbin loss induced epithelial-mesenchymal transition, which coincided with a significant increase in cell migration and invasion. Erbin interacted with kinase suppressor of Ras 1 (KSR1) and displaced it from the RAF/MEK/ERK complex to prevent signal propagation. Furthermore, genetic deletion of Erbin in Apc knockout mice promoted tumorigenesis and significantly reduced survival. Tumor organoids derived from Erbin/Apc double knockout mice displayed increased tumor initiation potential and activation of Wnt signaling. Results from gene set enrichment analysis revealed that Erbin expression associated positively with the E-cadherin adherens junction pathway and negatively with Wnt signaling in human colorectal cancer. Taken together, our study identifies Erbin as a negative regulator of tumor initiation and progression by suppressing Akt and RAS/RAF signaling in vivoSignificance: These findings establish the scaffold protein Erbin as a negative regulator of EMT and tumorigenesis in colorectal cancer through direct suppression of Akt and RAS/RAF signaling. Cancer Res; 78(17); 4839-52. ©2018 AACR.

PHLPP regulates hexokinase 2-dependent glucose metabolism in colon cancer cells.

Increased glucose metabolism is considered as one of the most important metabolic alterations adapted by cancer cells in order to generate energy as well as high levels of glycolytic intermediates to support rapid proliferation. PH domain leucine-rich repeat protein phosphatase (PHLPP) belongs to a novel family of Ser/Thr protein phosphatases that function as tumor suppressors in various types of human cancer. Here we determined the role of PHLPP in regulating glucose metabolism in colon cancer cells. Knockdown of PHLPP increased the rate of glucose consumption and lactate production, whereas overexpression of PHLPP had the opposite effect. Bioenergetic analysis using Seahorse Extracelluar Flux Analyzer revealed that silencing PHLPP expression induced a glycolytic shift in colon cancer cells. Mechanistically, we found that PHLPP formed a complex with Akt and hexokinase 2 (HK2) in the mitochondrial fraction of colon cancer cells and knockdown of PHLPP enhanced Akt-mediated phosphorylation and mitochondrial localization of HK2. Depletion of HK2 expression or treating cells with Akt and HK2 inhibitors reversed PHLPP loss-induced increase in glycolysis. Furthermore, PHLPP knockdown cells became addicted to glucose as a major energy source in that glucose starvation significantly decreased cancer cell survival. As HK2 is the key enzyme that determines the direction and magnitude of glucose flux, our study identified PHLPP as a novel regulator of glucose metabolism by controlling HK2 activity in colon cancer cells.

Adipocytes activate mitochondrial fatty acid oxidation and autophagy to promote tumor growth in colon cancer.

Obesity has been associated with increased incidence and mortality of a wide variety of human cancers including colorectal cancer. However, the molecular mechanism by which adipocytes regulate the metabolism of colon cancer cells remains elusive. In this study, we showed that adipocytes isolated from adipose tissues of colon cancer patients have an important role in modulating cellular metabolism to support tumor growth and survival. Abundant adipocytes were found in close association with invasive tumor cells in colon cancer patients. Co-culture of adipocytes with colon cancer cells led to a transfer of free fatty acids that released from the adipocytes to the cancer cells. Uptake of fatty acids allowed the cancer cells to survive nutrient deprivation conditions by upregulating mitochondrial fatty acid ß-oxidation. Mechanistically, co-culture of adipocytes or treating cells with fatty acids induced autophagy in colon cancer cells as a result of AMPK activation. Inhibition of autophagy attenuated the ability of cancer cells to utilize fatty acids and blocked the growth-promoting effect of adipocytes. In addition, we found that adipocytes stimulated the expression of genes associated with cancer stem cells and downregulated genes associated with intestinal epithelial cell differentiation in primary colon cancer cells and mouse tumor organoids. Importantly, the presence of adipocytes promoted the growth of xenograft tumors in vivo. Taken together, our results show that adipocytes in the tumor microenvironment serve as an energy provider and a metabolic regulator to promote the growth and survival of colon cancer cells.

PHLPP negatively regulates cell motility through inhibition of Akt activity and integrin expression in pancreatic cancer cells.

Pancreatic adenocarcinoma is currently the fourth leading cause for cancer-related mortality. Malignant progression of pancreatic cancer depends not only on rapid proliferation of tumor cells but also on increased cell motility. In this study, we showed that increased PHLPP expression significantly reduced the rate of migration in pancreatic ductal adenocarcinoma (PDAC) cells whereas knockdown of PHLPP had the opposite effect. In addition, cell motility at the individual cell level was negatively regulated by PHLPP as determined using time-lapse imaging. Interestingly, the expression of ß1 and ß4 integrin proteins were decreased in PHLPP overexpressing cells and increased in PHLPP knockdown cells whereas the mRNA levels of integrin were not altered by changes in PHLPP expression. In determining the molecular mechanism underlying PHLPP-mediated regulation of integrin expression, we found that inhibition of lysosome activity rescued integrin expression in PHLPP overexpressing cells, thus suggesting that PHLPP negatively controls cell motility by inhibiting Akt activity to promote lysosome-dependent degradation of integrins. Functionally, the increased cell migration observed in PHLPP knockdown cells was effectively blocked by the neutralizing antibodies against ß1 or ß4 integrin. Taken together, our study identified a tumor suppressor role of PHLPP in suppressing cell motility by negatively regulating integrin expression in pancreatic cancer cells.

Downregulation of PHLPP expression contributes to hypoxia-induced resistance to chemotherapy in colon cancer cells.

Hypoxia is a feature of solid tumors. Most tumors are at least partially hypoxic. This hypoxic environment plays a critical role in promoting resistance to anticancer drugs. PHLPP, a novel family of Ser/Thr protein phosphatases, functions as a tumor suppressor in colon cancers. Here, we show that the expression of both PHLPP isoforms is negatively regulated by hypoxia/anoxia in colon cancer cells. Interestingly, a hypoxia-induced decrease of PHLPP expression is attenuated by knocking down HIF1α but not HIF2α. Whereas the mRNA levels of PHLPP are not significantly altered by oxygen deprivation, the reduction of PHLPP expression is caused by decreased protein translation downstream of mTOR and increased degradation. Specifically, hypoxia-induced downregulation of PHLPP is partially rescued in TSC2 or 4E-BP1 knockdown cells as the result of elevated mTOR activity and protein synthesis. Moreover, oxygen deprivation destabilizes PHLPP protein by decreasing the expression of USP46, a deubiquitinase of PHLPP. Functionally, downregulation of PHLPP contributes to hypoxia-induced chemoresistance in colon cancer cells. Taken together, we have identified hypoxia as a novel mechanism by which PHLPP is downregulated in colon cancer, and the expression of PHLPP may serve as a biomarker for better understanding of chemoresistance in cancer treatment.

Silencing of PCDH10 in hepatocellular carcinoma via de novo DNA methylation independent of HBV infection or HBX expression.

PCDH10 is a key tumor suppressive gene for nasopharyngeal, esophageal, and other carcinomas with frequent methylation. In this study, we investigated the potential epigenetic modification of the PCDH10 gene by hepatitis B virus × protein (HBx), a pivotal factor in the progression of HBV replication and potential carcinogenesis. PCDH10 expression was found to be down-regulated in 9/13 (69.2 %) of hepatocellular carcinoma (HCC) cell lines. Decreased PCDH10 expression was correlated with the methylation status of the PCDH10 promoter. Treatment with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (Aza) was sufficient to restore PCDH10 mRNA expression by suppressing PCDH10 promoter methylation in HepG2 cells. Treatment with Trichostatin A alone had no significant effect on PCDH10 expression but enhanced the effect of Aza. PCDH10 methylation was further detected in 76 % (38 of 50) of HCC tissues compared with 40 % (20 of 50) of paired adjacent tissues, with no methylation detected in normal human liver tissues. There were significant correlations between methylation status of PCDH10 and tumor size, serum AFP levels, metastasis or TNM staging (P < 0.05). Moreover, PCDH10 promoter methylation status was not associated with HBV infection in our panel of 50 primary HCC tumors, and transfection with HBX could not alter the status of PCDH10 promoter methylation. Collectively, these observations suggested that the expression of PCDH10 was silenced in HCC via de novo DNA methylation independent of HBV infection or HBX expression, and PCDH10 might form a potentially useful therapeutic target for HCC.

Use of duplex mutation primers for real-time PCR quantification of hepatitis C virus RNA in serum.

BACKGROUND: The duplex mutation primers offer many advantages over other multi-labeled probes for real-time detection of amplification products. OBJECTIVES: To develop and validate a novel real-time PCR for quantification of HCV RNA based on the duplex mutation primers technology. MATERIALS AND METHODS: The duplex mutation primers were selected in the highly conservative 5' non-coding region (5'NCR) of the HCV RNA. The assay was validated with the Viral Quality Control panel, which also includes Chinese HCV RNA standards. RESULTS: The detection limit was 57 IU/mL, and a good linear correlation in the range of 102-108 IU/mL was revealed (r(2) = 0.999) with the novel method. This assay has a dynamic range of at least 8 log10 without the need for specimen dilution, good clinical intra- and inter-run precision, and excellent correlation with a commercially available assay(r(2) = 0.95). CONCLUSIONS: The high sensitivity, wide linear range, and good reproducibility, combined with low cost, make this novel quantitative HCV real-time PCR assay particularly well suited for application to clinical and epidemiological studies.

Biliary intervention aggravates cholestatic liver injury, and induces hepatic inflammation, proliferation and fibrogenesis in BDL mice.

Obstructive cholestasis occurs in various clinical situations, whose pathological process is complex and not well known. The present study was initiated to display the complex and multifaceted pathological process caused by obstructive cholestasis in bile duct-ligated mice. Adult mice were bile-duct-ligated or sham-operated, and serum and liver tissues were collected at the indicated time points. Automatic biochemical analyzer was used to monitor serum biochemical index; TUNEL, HE staining, immunohistochemistry and Real-time PCR were employed to evaluate liver apoptosis, necrosis, inflammation, as well as proliferation and fibrosis. Our results demonstrated that obstructive cholestasis led to elevated serum biochemical indicators, with ALT peaking at day 3, indicative of acute hepatic dysfunction. Meanwhile, the number of TUNEL-positive cells increased significantly, and by 2 weeks, mild to moderate necrosis became apparent in BDL mouse livers, which consequently aggravated hepatic inflammatory responses as was demonstrated by increased expression of KC-1, MIP-2, ICAM-1 and MPO in BDL mouse livers. Moreover, proliferative hepatocytes around periportal areas, manifested by enhanced cell mitosis and elevated expression of proliferative markers such as PCNA and Ki67, increased significantly after BDL, while increased CK-19-positive cells in bile ducts indicated bile duct hyperplasia. By 2 weeks, numerous α-SMA-positive cells and Sirius-stained collagen were observed, indicative of hepatic stellate cells (HSC) activation and fibrogenesis. In conclusion, biliary intervention led to a multifaceted hepatic pathological process characterized by aggravated liver injury and inflammatory reaction with enhanced cellular proliferation and fibrogenesis.

C/EBPα down-regulation is associated with reduced hepatic cellular viability during hypoxia in vitro and in vivo.

C/EBPα transcription factor is a key regulator in liver biology and was preliminarily shown to be down-regulated in hypoxic primary rat hepatocytes. The aim of this study was to explore the possible association between C/EBPα expression level and hepatocyte viability in both the in-vitro cultured hypoxic rat primary hepatocytes and two models of acute liver hypoxia induced by carbon tetrachloride or Fas antibody. C/EBPα mRNA was significantly down-regulated under hypoxic conditions both in vitro and in vivo, which was paralleled by a similar decrease in hepatocyte viability and partially reversed by 3D matrix and dexamethasone. These results suggested that C/EBPα down-regulation may be one mechanism of reduced hepatocyte viability in these settings.