Wendy M. Mars

Hepatocyte growth factor/scatter factor is a motogen for interneurons migrating from the ventral to dorsal telencephalon.

Cortical interneurons arise from the proliferative zone of the ventral telencephalon, the ganglionic eminence, and migrate into the developing neocortex. The spatial patterns of migratory interneurons reflect the complementary expression of hepatocyte growth factor/scatter factor (HGF/SF) and its receptor, MET, in the forebrain. Scatter assays on forebrain explants demonstrate regionally specific motogenic activity due to HGF/SF. In addition, exogenous ligand disrupts normal cell migration. Mice lacking the urokinase-type plasminogen activator receptor (u-PAR), a key component of HGF/SF activation, exhibit deficient scatter activity in the forebrain, abnormal interneuron migration from the ganglionic eminence, and reduced interneurons in the frontal and parietal cortex. The data suggest that HGF/SF motogenic activity, which is essential for normal development of other organ systems, is a conserved mechanism that regulates trans-telencephalic migration of interneurons.

Disruption of tissue-type plasminogen activator gene in mice reduces renal interstitial fibrosis in obstructive nephropathy

Tissue-type plasminogen activator (tPA) is one of the major components in the matrix proteolytic network whose role in the pathogenesis of renal interstitial fibrosis remains largely unknown. Here, we demonstrate that ablation of tPA attenuated renal interstitial fibrotic lesions in obstructive nephropathy. Mice lacking tPA developed less morphological injury and displayed a reduced deposition of interstitial collagen III and fibronectin as well as total tissue collagen in the kidneys after sustained ureteral obstruction, when compared with their wild-type counterparts. Deficiency of tPA selectively blocked tubular epithelial-to-myofibroblast transition (EMT), but did not affect myofibroblastic activation from interstitial fibroblasts. A marked decrease in matrix metalloproteinase-9 (MMP-9) induction was found in the obstructed kidneys of tPA(-/-) mice, which led to a dramatic preservation of the structural and functional integrity of tubular basement membrane (TBM). In vitro, tPA induced MMP-9 gene expression and protein secretion in renal interstitial fibroblasts. Thus, increased tPA is detrimental in renal interstitial fibrogenesis through a cascade of events that lead to MMP-9 induction, TBM destruction, and promotion of EMT. Our findings establish a crucial and definite importance of EMT in the pathogenesis of renal interstitial fibrosis at the whole-animal level.

Tissue-type plasminogen activator acts as a cytokine that triggers intracellular signal transduction and induces matrix metalloproteinase-9 gene expression.

Tissue-type plasminogen activator (tPA), a serine protease well known for generating plasmin, has been demonstrated to induce matrix metalloproteinase-9 (MMP-9) gene expression and protein secretion in renal interstitial fibroblasts. However, exactly how tPA transduces its signal into the nucleus to control gene expression is unknown. This study investigated the mechanism by which tPA induces MMP-9 gene expression. Both wild-type and non-enzymatic mutant tPA were found to induce MMP-9 expression in rat kidney interstitial fibroblasts (NRK-49F), indicating that the actions of tPA are independent of its proteolytic activity. tPA bound to the low density lipoprotein receptor-related protein-1 (LRP-1) in NRK-49F cells, and this binding was competitively abrogated by the LRP-1 antagonist, the receptor-associated protein. In mouse embryonic fibroblasts (PEA-13) lacking LRP-1, tPA failed to induce MMP-9 expression. Furthermore, tPA induced rapid tyrosine phosphorylation on the β subunit of LRP-1, which was followed by the activation of Mek1 and its downstream Erk-1 and -2. Blockade of Erk-1/2 activation by the Mek1 inhibitor abolished MMP-9 induction by tPA in NRK-49F cells. Conversely, overexpression of constitutively activated Mek1 induced Erk-1/2 phosphorylation and MMP-9 expression. In mouse obstructed kidney, tPA, LRP-1, and MMP-9 were concomitantly induced in the renal interstitium. Collectively, these results suggest that besides its classical proteolytic activity, tPA acts as a cytokine that binds to the cell membrane receptor LRP-1, induces its tyrosine phosphorylation, and triggers intracellular signal transduction, thereby inducing specific gene expression in renal interstitial fibroblasts.

Enhanced liver regeneration following changes induced by hepatocyte-specific genetic ablation of integrin-linked kinase

Following liver regeneration after partial hepatectomy, liver grows back precisely to its original mass and does not exceed it. The mechanism regulating this “hepatostat” is not clear and no exceptions have been found to date. Although pathways initiating liver regeneration have been well studied, mechanisms involved in the termination of liver regeneration are unclear. Here, we report that integrin‐linked kinase (ILK) (involved in transmission of the extracellular matrix [ECM] signaling by way of integrin receptors) and/or hepatic adaptations that ensue following ILK hepatocyte‐targeted removal are critical for proper termination of liver regeneration. Following partial hepatectomy (PHx), mice with a liver‐specific ILK ablation (ILK‐KO‐Liver) demonstrate a termination defect resulting in 58% larger liver than their original pre‐PHx mass. This increase in post‐PHx liver mass is due to sustained cell proliferation driven in part by increased signaling through hepatocyte growth factor (HGF), and the β‐catenin pathway and Hippo kinase pathways. Conclusion: The data indicate that ECM‐mediated signaling by way of ILK is essential in proper termination of liver regeneration. This is the first evidence of a defect leading to impaired termination of regeneration and excessive accumulation of liver weight following partial hepatectomy. (HEPATOLOGY 2009.)

Regulation of Rac1 activation by the low density lipoprotein receptor-related protein.

The low density lipoprotein receptor–related protein (LRP-1) binds and mediates the endocytosis of multiple ligands, transports the urokinase-type plasminogen activator receptor (uPAR) and other membrane proteins into endosomes, and binds intracellular adaptor proteins involved in cell signaling. In this paper, we show that in murine embryonic fibroblasts (MEFs) and L929 cells, LRP-1 functions as a major regulator of Rac1 activation, and that this activity depends on uPAR. LRP-1–deficient MEFs demonstrated increased Rac1 activation compared with LRP-1–expressing MEFs, and this property was reversed by expressing the VLDL receptor, a member of the same gene family as LRP-1, with overlapping ligand-binding specificity. Neutralizing the activity of LRP-1 with receptor-associated protein (RAP) increased Rac1 activation and cell migration in MEFs and L929 cells. The same parameters were unaffected by RAP in uPAR−/− MEFs, prepared from uPAR gene knockout embryos, and in uPAR-deficient LM-TK− cells. Untreated uPAR+/+ MEFs demonstrated substantially increased Rac1 activation compared with uPAR−/− MEFs. In addition to Rac1, LRP-1 suppressed activation of extracellular signal–regulated kinase (ERK) in MEFs; however, it was Rac1 (and not ERK) that was responsible for the effects of LRP-1 on MEF migration. Thus, LRP-1 regulates two signaling proteins in the same cell (Rac1 and ERK), both of which may impact on cell migration. In uPAR-negative cells, LRP-1 neutralization does not affect Rac1 activation, and other mechanisms by which LRP-1 may regulate cell migration are not unmasked.

Tissue-type plasminogen activator promotes murine myofibroblast activation through LDL receptor–related protein 1–mediated integrin signaling

The activation of interstitial fibroblasts to become alpha-SMA-positive myofibroblasts is an essential step in the evolution of chronic kidney fibrosis, as myofibroblasts are responsible for the production and deposition of the ECM components that are a hallmark of the disease. Here we describe a signaling pathway that leads to this activation. Tissue-type plasminogen activator (tPA) promoted TGF-beta1-mediated alpha-SMA and type I collagen expression in rat kidney interstitial fibroblasts. This fibrogenic effect was independent of its protease activity but required its membrane receptor, the LDL receptor-related protein 1 (LRP-1). In rat kidney fibroblasts, tPA induced rapid LRP-1 tyrosine phosphorylation and enhanced beta1 integrin recruitment by facilitating the LRP-1/beta1 integrin complex formation. Blockade or knockdown of beta1 integrin abolished type I collagen and alpha-SMA expression. Furthermore, inhibition of the integrin-linked kinase (ILK), a downstream effector of beta1 integrin, or disruption of beta1 integrin/ILK engagement, abrogated the tPA action, whereas ectopic expression of ILK mimicked tPA in promoting myofibroblast activation. In murine renal interstitium after obstructive injury, tPA and alpha-SMA colocalized with LRP-1, and tPA deficiency reduced LRP-1/beta1 integrin interaction and myofibroblast activation. These findings show that tPA induces LRP-1 tyrosine phosphorylation, which in turn facilitates the LRP-1-mediated recruitment of beta1 integrin and downstream ILK signaling, thereby leading to myofibroblast activation. This study implicates tPA as a fibrogenic cytokine that promotes the progression of kidney fibrosis.

Hepatocyte Growth Factor Modulates Interleukin-6 Production in Bone Marrow Derived Macrophages: Implications for Inflammatory Mediated Diseases

The generation of the pro-inflammatory cytokines IL-6, TNF-α, and IL-1β fuel the acute phase response (APR). To maintain body homeostasis, the increase of inflammatory proteins is resolved by acute phase proteins via presently unknown mechanisms. Hepatocyte growth factor (HGF) is transcribed in response to IL-6. Since IL-6 production promotes the generation of HGF and induces the APR, we posited that accumulating HGF might be a likely candidate for quelling excess inflammation under non-pathological conditions. We sought to assess the role of HGF and how it influences the regulation of inflammation utilizing a well-defined model of inflammatory activation, lipopolysaccharide (LPS)-stimulation of bone marrow derived macrophages (BMM). BMM were isolated from C57BL6 mice and were stimulated with LPS in the presence or absence of HGF. When HGF was present, there was a decrease in production of the pro-inflammatory cytokine IL-6, along with an increase in the anti-inflammatory cytokine IL-10. Altered cytokine production correlated with an increase in phosphorylated GSK3β, increased retention of the phosphorylated NFκB p65 subunit in the cytoplasm, and an enhanced interaction between CBP and phospho-CREB. These changes were a direct result of signaling through the HGF receptor, MET, as effects were reversed in the presence of a selective inhibitor of MET (SU11274) or when using BMM from macrophage-specific conditional MET knockout mice. Combined, these data provide compelling evidence that under normal circumstances, HGF acts to suppress the inflammatory response.

Liver‐specific ablation of integrin‐linked kinase in mice results in abnormal histology, enhanced cell proliferation, and hepatomegaly

Hepatocyte differentiation and proliferation are greatly affected by extracellular matrix (ECM). Primary hepatocytes cultured without matrix dedifferentiate over time, but matrix overlay quickly restores differentiation. ECM also is critical in liver regeneration where ECM degradation and reconstitution are steps in the regenerative process. Integrin‐linked kinase (ILK) is a cell‐ECM‐adhesion component implicated in cell–ECM signaling by means of integrins. We investigated the role of ILK in whole liver by using the LoxP/Cre model system. ILK was eliminated from the liver by mating homozygous ILK‐floxed animals with mice expressing Cre‐recombinase under control of the α fetoprotein enhancer and albumin promoter. After ablation of ILK, animals are born normal. Soon after birth, however, they develop histologic abnormalities characterized by disorderly hepatic plates, increased proliferation of hepatocytes and biliary cells, and increased deposition of extracellular matrix. Cell proliferation is accompanied by increased cytoplasmic and nuclear stabilization of β‐catenin. After this transient proliferation of all epithelial components, proliferation subsides and final liver to body weight ratio in livers with ILK deficient hepatocytes is two times that of wild type. Microarray analysis of gene expression during the stage of cell proliferation shows up‐regulation of integrin and matrix‐related genes and a concurrent down‐regulation of differentiation‐related genes. After the proliferative stage, however, the previous trends are reversed resulting in a super‐differentiated phenotype in the ILK‐deficient livers. Conclusion: Our results show for the first time in vivo the significance of ILK and hepatic ECM‐signaling for regulation of hepatocyte proliferation and differentiation. (HEPATOLOGY 2008;48:1932‐1941.)

Cationic Colloidal Silica Membrane Perturbation as a Means of Examining Changes at the Sinusoidal Surface during Liver Regeneration

By employing the cationic colloidal silica membrane density perturbation technique, we examined growth factor receptor and extracellular matrix (ECM) changes at the sinusoidal surface during rat liver regeneration 72 hours after 70% partial hepatectomy (PHx). At this time after PHx, hepatocyte division has mostly subsided, while sinusoidal endothelial cell (SEC) proliferation is initiating, resulting in avascular hepatocyte islands. Because of the discontinuous nature of the surface of liver SEC, ECM proteins underlying the SEC, as well as SEC luminal membrane proteins, are available to absorption to the charged silica beads when the liver is perfused with the colloid. Subsequent liver homogenization and density centrifugation yield two separate fractions, enriched in SECs as well as hepatocyte basolateral membrane-specific proteins up to 50-fold over whole liver lysates. This technique facilitates examination of changes in protein composition that influence or occur as a result of SEC mitogenesis and migration during regeneration of the liver. When ECM and receptor proteins from SEC-enriched fractions were examined by Western immunoblotting, urokinase plasminogen activator receptor, fibronectin, and plasmin increased at the SEC surface 72 hours after PHx. Epidermal growth factor receptor, plasminogen, SPARC (secreted protein, acidic and rich in cysteine, also called osteonectin or BM40), and collagen IV decreased, and fibrinogen subunits and c-Met expression remained constant 72 hours after PHx when compared to control liver. These results display the usefulness of the cationic colloidal silica membrane isolation protocol. They also show considerable modulation of surface components that may regulate angiogenic processes at the end stage of liver regeneration during the reformation of sinusoids.

Signals and Cells Involved in Regulating Liver Regeneration

Liver regeneration is a complex phenomenon aimed at maintaining a constant liver mass in the event of injury resulting in loss of hepatic parenchyma. Partial hepatectomy is followed by a series of events involving multiple signaling pathways controlled by mitogenic growth factors (HGF, EGF) and their receptors (MET and EGFR). In addition multiple cytokines and other signaling molecules contribute to the orchestration of a signal which drives hepatocytes into DNA synthesis. The other cell types of the liver receive and transmit to hepatocytes complex signals so that, in the end of the regenerative process, complete hepatic tissue is assembled and regeneration is terminated at the proper time and at the right liver size. If hepatocytes fail to participate in this process, the biliary compartment is mobilized to generate populations of progenitor cells which transdifferentiate into hepatocytes and restore liver size.