Renay L. Bauer

Proinflammatory Cytokine Production in Liver Regeneration Is Myd88-Dependent, but Independent of Cd14, Tlr2, and Tlr4

TNF and IL-6 are considered to be important to the initiation or priming phase of liver regeneration. However, the signaling pathways that lead to the production of these cytokines after partial hepatectomy (PH) have not been identified. Enteric-derived LPS appears to be important to liver regeneration, possibly by stimulating proinflammatory cytokine production after surgery. To determine whether LPS signaling pathways are involved in the regulation of the proinflammatory cytokines TNF and IL-6 during the priming phase of liver regeneration, we performed PH on mice lacking the TLRs Tlr4 and Tlr2, the LPS coreceptor, Cd14, and Myd88, an adapter protein involved in most TLR and IL-1R pathways. In MyD88 knockout (KO) mice after PH, both liver Tnf mRNA and circulating IL-6 levels were severely depressed compared with heterozygous or wild-type mice. Activation of STAT-3 and three STAT-3 responsive genes, Socs3, Cd14, and serum amyloid A2 were also blocked. In contrast, Tlr4, Tlr2, and Cd14 KO mice showed no deficits in the production of IL-6. Surprisingly, none of these KO mice showed any delay in hepatocyte replication. These data indicate that the LPS receptor TLR4, as well as TLR2 and CD14, do not play roles in regulating cytokine production or DNA replication after PH. In contrast, MyD88-dependent pathways appear to be responsible for TNF, IL-6, and their downstream signaling pathways.

Activation of platelet-derived growth factor receptor alpha contributes to liver fibrosis.

Chronic liver injury leads to fibrosis, cirrhosis, and loss of liver function. Liver cirrhosis is the 12th leading cause of death in the United States, and it is the primary risk factor for developing liver cancer. Fibrosis and cirrhosis result from activation of hepatic stellate cells (HSCs), which are the primary collagen producing cell type in the liver. Here, we show that platelet-derived growth factor receptor α (PDGFRα) is expressed by human HSCs, and PDGFRα expression is elevated in human liver disease. Using a green fluorescent protein (GFP) reporter mouse strain, we evaluated the role of PDGFRα in liver disease in mice and found that mouse HSCs express PDGFRα and expression is upregulated during carbon tetrachloride (CCl4) induced liver injury and fibrosis injection. This fibrotic response is reduced in Pdgfrα heterozygous mice, consistent with the hypothesis that liver fibrosis requires upregulation and activation of PDGFRα. These results indicate that Pdgfrα expression is important in the fibrotic response to liver injury in humans and mice, and suggest that blocking PDGFRα–specific signaling pathways in HSCs may provide therapeutic benefit for patients with chronic liver disease.

Toll‐like receptor 4 and myeloid differentiation factor 88 provide mechanistic insights into the cause and effects of interleukin‐6 activation in mouse liver regeneration

Partial hepatectomy (PH) consistently results in an early increase of circulating interleukin‐6 (IL‐6), which is thought to play a major role in liver regeneration. Activation of this cytokine after PH requires the adaptor protein, MyD88, but the specific MyD88‐related receptors involved remain unidentified. It is also unknown whether the magnitude of IL‐6 elevation determines the extent of subsequent hepatocyte proliferation. Here, we uncovered artifacts in the assessment of circulating IL‐6 levels when using cardiac puncture in mice after PH. By using retro‐orbital bleed sampling, we show that the circulating levels of IL‐6 after PH were not directly correlated with the extent of hepatocyte DNA synthesis in individual mice. The IL‐6 increase after PH was attenuated in all lipopolysaccharide‐hyporesponsive mouse strains studied (e.g., C3H/HeJ, Tlr4 null, Cd14 null, Tlr2,4,9 null, and Tlr2,4‐Caspase1 null) and was severely abrogated in Myd88 null mice. Despite attenuated IL‐6 levels, Tlr4 null mice showed normal signaling downstream of IL‐6 and normal hepatocyte proliferation. In contrast, Myd88 null mice showed severe impairments in signal transducer and activator of transcription 3 phosphorylation and Socs3 induction, but had enhanced and prolonged extracellular signal‐related kinase 1 and 2 phosphorylation in the first 6 hours after PH. Unexpectedly, these changes were associated with accelerated initiation of hepatocyte proliferation, as assessed by hepatocyte bromodeoxyuridine incorporation, phospho‐histone H3 immunostaining, and cyclin E and A protein expression. Conclusion: TLR‐4 signaling contributes to IL‐6 activation after PH, but the Tlr4‐independent component appears sufficient for ensuring intact signaling downstream of IL‐6. The lack of correlation between IL‐6 levels and hepatocyte proliferation after PH, and the accelerated start of hepatocyte proliferation in Myd88 null mice despite abrogated cytokine activation, may highlight relevant antiproliferative effects of IL‐6 signaling, possibly via Socs3, in the regulation of liver regeneration. (HEPATOLOGY 2011;)

Paracrine activation of hepatic stellate cells in platelet-derived growth factor C transgenic mice: evidence for stromal induction of hepatocellular carcinoma.

Cirrhosis is the primary risk factor for the development of hepatocellular carcinoma (HCC), yet the mechanisms by which cirrhosis predisposes to carcinogenesis are poorly understood. Using a mouse model that recapitulates many aspects of the pathophysiology of human liver disease, we explored the mechanisms by which changes in the liver microenvironment induce dysplasia and HCC. Hepatic expression of platelet‐derived growth factor C (PDGF‐C) induces progressive fibrosis, chronic inflammation, neoangiogenesis and sinusoidal congestion, as well as global changes in gene expression. Using reporter mice, immunofluorescence, immunohistochemistry and liver cell isolation, we demonstrate that receptors for PDGF‐CC are localized on hepatic stellate cells (HSCs), which proliferate, and transform into myofibroblast‐like cells that deposit extracellular matrix and lead to production of growth factors and cytokines. We demonstrate induction of cytokine genes at 2 months, and stromal cell‐derived hepatocyte growth factors that coincide with the onset of dysplasia at 4 months. Our results support a paracrine signaling model wherein hepatocyte‐derived PDGF‐C stimulates widespread HSC activation throughout the liver leading to chronic inflammation, liver injury and architectural changes. These complex changes to the liver microenvironment precede the development of HCC. Further, increased PDGF‐CC levels were observed in livers of patients with nonalcoholic fatty steatohepatitis and correlate with the stage of disease, suggesting a role for this growth factor in chronic liver disease in humans. PDGF‐C transgenic mice provide a unique model for the in vivo study of tumor–stromal interactions in the liver.

Role of Smad3 in Platelet-Derived Growth Factor-C induced liver fibrosis

Chronic liver injury leads to fibrosis and cirrhosis. Cirrhosis, the end stage of chronic liver disease, is a leading cause of death worldwide and increases the risk of developing hepatocellular carcinoma. Currently, there is a lack of effective antifibrotic therapies to treat fibrosis and cirrhosis. Development of antifibrotic therapies requires an in-depth understanding of the cellular and molecular mechanisms involved in inflammation and fibrosis after hepatic injury. Two growth factor signaling pathways that regulate liver fibrosis are transforming growth factor-β (TGFβ) and platelet-derived growth factor (PDGF). However, their specific contributions to fibrogenesis are not well understood. Using a genetic model of liver fibrosis, we investigated whether the canonical TGFβ signaling pathway was necessary for fibrogenesis. PDGF-C transgenic (PDGF-C Tg) mice were intercrossed with mice that lack Smad3, and molecular and histological fibrosis was analyzed. PDGF-C Tg mice that also lacked Smad3 had less fibrosis and improved liver lobule architecture. Loss of Smad3 also reduced expression of collagen genes, which were induced by PDGF-C, but not the expression of genes frequently associated with hepatic stellate cell (HSC) activation. In vitro HSCs isolated from Smad3-null mice proliferated more slowly than cells from wild-type mice. Taken together, these findings indicate that PDGF-C activates TGFβ/Smad3 signaling pathways to regulate HSC proliferation, collagen production and ultimately fibrosis. In summary, these results suggest that inhibition of both PDGF and TGFβ signaling pathways may be required to effectively attenuate fibrogenesis in patients with chronic liver disease.

Enhanced cAMP-stimulated protein kinase A activity in human fibrolamellar hepatocellular carcinoma.

Background:Fibrolamellar hepatocellular carcinoma (FL-HCC) affects children without underlying liver disease. A consistent mutation in FL-HCCs leads to fusion of the genes encoding a heat shock protein (DNAJB1) and the catalytic subunit of protein kinase A (PRKACA). We sought to characterize the resultant chimeric protein and its effects in FL-HCC.Methods:The expression pattern and subcellular localization of protein kinase A (PKA) subunits in FL-HCCs were compared to paired normal livers by quantitative polymerase chain reaction (qPCR), immunoblotting, and immunofluorescence. PKA activity was measured by radioactive kinase assay, and we determined whether the FL-HCC mutation is present in other primary liver tumors.Results:The fusion transcript and chimeric protein were detected exclusively in FL-HCCs. DNAJB1-PRKACA was expressed 10-fold higher than the wild-type PRKACA transcript, resulting in overexpression of the mutant protein in tumors. Consequently, FL-HCCs possess elevated cAMP-stimulated PKA activity compared to normal livers, despite similar Kms between the mutant and wild-type kinases.Conclusion:FL-HCCs in children and young adults uniquely overexpress DNAJB1-PRKACA, which results in elevated cAMP-dependent PKA activity. These data suggest that aberrant PKA signaling contributes to liver tumorigenesis.

Tissue-type plasminogen activator is not necessary for platelet-derived growth factor-c activation

Platelet-derived growth factors (PDGFs) are critical for development; their over-expression is associated with fibrogenesis. Full-length PDGF-C is secreted as an inactive dimer, requiring cleavage to allow receptor binding. Previous studies indicate that tissue-type plasminogen activator (tPA) is the specific protease that performs this cleavage; in vivo confirmation is lacking. We demonstrate that primary hepatocytes from tpa KO mice produce less cleaved active PDGF-CC than do wild type hepatocytes, suggesting that tPA is critical for in vitro activation of this growth factor. We developed mice that over-express full-length human PDGF-C in the liver; these mice develop progressive liver fibrosis. To test whether tPA is important for cleavage and activation of PDGF-C in vivo, we intercrossed PDGF-C transgenic (Tg) and tpa knock-out (KO) mice, anticipating that lack of tPA would result in decreased fibrosis due to lack of hPDGF-C cleavage. To measure levels of cleaved, dimerized PDGF-CC in sera, we developed an ELISA that specifically detects cleaved PDGF-CC. We report that the absence of tpa does not affect the phenotype of `PDGF-C Tg mice. PDGF-C Tg mice lacking tPA have high serum levels of cleaved growth factor, significant liver fibrosis, and gene expression alterations similar to those of PDGF-C Tg mice with intact tPA. Furthermore, urokinase plasminogen activator and plasminogen activator inhibitor-1 expression are increased in PDGF-C Tg; tpa KO mice. Our ELISA data suggest a difference between in vitro and in vivo activation of this growth factor, and our mouse model confirms that multiple proteases cleave and activate PDGF-C in vivo.

Characterization of a novel mutation in fibrolamellar hepatocellular carcinoma.

284 Background: Fibrolamellar hepatocellular carcinoma (FL-HCC) is a subtype of HCC occurring in children and young adults in the absence of underlying liver disease. Currently, there is no effective therapy for unresectable or metastatic FL-HCC. Recent genomic analysis identified a consistent mutation in FL-HCC involving a deletion on chromosome 19 resulting in a chimeric transcript containing the 5’-region of a heat shock protein (DNAJB1) fused to the catalytic subunit of protein kinase A (PRKACA). We sought to characterize the resultant protein and its effects on PKA activity in human FL-HCC. Methods: We prepared tissue lysates from four snap-frozen FL-HCC samples with paired, non-tumor liver as well as adult HCCs. PKA activity was determined via a radioactive kinase assay in the presence and absence of cAMP, a PKA activator. RNA was extracted using TRIZOL, and used for qRT-PCR. Triple immuno-fluorescent labeling was performed using antibodies to PRKACA, PKA RIIα, and a nuclear marker, DRAQ5. Results: ...

Paracrine activation of hepatic stellate cells in platelet-derived growth factor C transgenic mice

Cirrhosis is the primary risk factor for the development of hepatocellular carcinoma (HCC), yet the mechanisms by which cirrhosis predisposes to carcinogenesis are poorly understood. Using a mouse model that recapitulates many aspects of the pathophysiology of human liver disease, we explored the mechanisms by which changes in the liver microenvironment induce dysplasia and HCC. Hepatic expression of platelet‐derived growth factor C (PDGF‐C) induces progressive fibrosis, chronic inflammation, neoangiogenesis and sinusoidal congestion, as well as global changes in gene expression. Using reporter mice, immunofluorescence, immunohistochemistry and liver cell isolation, we demonstrate that receptors for PDGF‐CC are localized on hepatic stellate cells (HSCs), which proliferate, and transform into myofibroblast‐like cells that deposit extracellular matrix and lead to production of growth factors and cytokines. We demonstrate induction of cytokine genes at 2 months, and stromal cell‐derived hepatocyte growth factors that coincide with the onset of dysplasia at 4 months. Our results support a paracrine signaling model wherein hepatocyte‐derived PDGF‐C stimulates widespread HSC activation throughout the liver leading to chronic inflammation, liver injury and architectural changes. These complex changes to the liver microenvironment precede the development of HCC. Further, increased PDGF‐CC levels were observed in livers of patients with nonalcoholic fatty steatohepatitis and correlate with the stage of disease, suggesting a role for this growth factor in chronic liver disease in humans. PDGF‐C transgenic mice provide a unique model for the in vivo study of tumor–stromal interactions in the liver.

Paracrine activation of hepatic stellate cells in platelet-derived growth factor C transgenic mice: Evidence for stromal induction of hepatocellular carcinoma: Stromal activation in PDGF-C transgenic mice

Cirrhosis is the primary risk factor for the development of hepatocellular carcinoma (HCC), yet the mechanisms by which cirrhosis predisposes to carcinogenesis are poorly understood. Using a mouse model that recapitulates many aspects of the pathophysiology of human liver disease, we explored the mechanisms by which changes in the liver microenvironment induce dysplasia and HCC. Hepatic expression of platelet‐derived growth factor C (PDGF‐C) induces progressive fibrosis, chronic inflammation, neoangiogenesis and sinusoidal congestion, as well as global changes in gene expression. Using reporter mice, immunofluorescence, immunohistochemistry and liver cell isolation, we demonstrate that receptors for PDGF‐CC are localized on hepatic stellate cells (HSCs), which proliferate, and transform into myofibroblast‐like cells that deposit extracellular matrix and lead to production of growth factors and cytokines. We demonstrate induction of cytokine genes at 2 months, and stromal cell‐derived hepatocyte growth factors that coincide with the onset of dysplasia at 4 months. Our results support a paracrine signaling model wherein hepatocyte‐derived PDGF‐C stimulates widespread HSC activation throughout the liver leading to chronic inflammation, liver injury and architectural changes. These complex changes to the liver microenvironment precede the development of HCC. Further, increased PDGF‐CC levels were observed in livers of patients with nonalcoholic fatty steatohepatitis and correlate with the stage of disease, suggesting a role for this growth factor in chronic liver disease in humans. PDGF‐C transgenic mice provide a unique model for the in vivo study of tumor–stromal interactions in the liver.