Activation of Rac1 promotes hedgehog-mediated acquisition of the myofibroblastic phenotype in rat and human hepatic stellate cells.

Chronic liver injury induces regenerative responses in the liver, including the activation of quiescent hepatic stellate cells (Q-HSC) to an activated, myofibroblastic phenotype that promotes scar formation and liver dysfunction.(1) Hepatic accumulation of myofibroblastic hepatic stellate cells (MF-HSC) is pivotal in the pathogenesis of cirrhosis. Therefore, considerable research has been directed towards delineating mechanisms that promote the transition of Q-HSC to MF-HSC, and that enhance the subsequent growth of MF-HSC populations.(2) Recently, we showed that Rac1 is one factor that modulates hepatic accumulation of MF-HSC. Rac1 belongs to a sub-family of small GTP-binding proteins that includes Ras-related C3 botulinum toxin substrate 1 (rac1), rac2, rac3, and several rac-related proteins.(3) Rac1 regulates many cellular processes, including the cell cycle, cell-cell adhesion, and motility.(3-6) It also maintains epidermal stem cells that generate epithelial tissues.(7) We demonstrated that Rac1 is activated as Q-HSC become MF-HSC.(8) Using a genetic approach to maintain high levels of Rac1 activity in MF-HSC, we showed that this enhanced MF-HSC growth in culture. We also demonstrated that transgenic mice expressing constitutively active human Rac1 in MF-HSC accumulated more myofibroblastic cells and developed worse liver fibrosis than littermates during liver injury.(8) These data established activated Rac1 as an important mediator of MF-HSC accumulation and fibrosis. The Hedgehog (Hh) pathway also modulates MF-HSC accumulation and liver fibrosis.(9, 10) Hh ligands are lipid-modified morphogens that interact with Patched (Ptc), a membrane-spanning receptor on Hh-responsive cells. This ligand-receptor interaction prevents Ptc from inhibiting its co-receptor, Smoothened (Smo). Smo, in turn, initiates a series of intracellular events that culminate in activation and nuclear localization of Glioblastoma (Gli)-family transcription factors.(11) Hh signaling is antagonized by hedgehog-interacting protein (Hhip), a factor that binds to Hh ligands and blocks Hh ligand-Ptc interactions.(12) We have shown that Hh signaling is involved in adult liver repair.(10, 13) Factors produced during liver injury stimulate MF-HSC to produce Sonic hedgehog (Shh) ligand.(9) Shh acts in an autocrine fashion to promote MF-HSC proliferation and viability,(9) and in a paracrine fashion to stimulate the viability of ductular-type progenitor cells (DPC).(13) Genetically-altered mice with an overly active Hh pathway accumulate more MF-HSC and develop worse liver fibrosis after bile duct ligation (BDL) than littermate controls.(13) Whether Hh signaling interacts with, or requires, Rac1 to increase MF-HSC remains unclear. Therefore, the primary objective of the current study was to evaluate the hypothesis that Rac1 and the Hh pathway interact to promote the formation of MF-HSC and/or to enhance accumulation of existent MF-HSC. A secondary aim was to begin to characterize mechanisms involved in these processes. During development, adult wound healing, and cancer metastasis, the Hh pathway is known to promote formation of mesenchymal cell types by inducing epithelial-to-mesenchymal transitions (EMT).(14, 15) EMT is a process that permits tissue remodeling by repressing expression of adherens junction-proteins (e.g., E-cadherin), reducing cell-cell adhesion, and promoting cell motility.(16) EMT may contribute to liver fibrosis.(17) It was recently reported that treating MF-HSC with adenoviral vectors for bone morphogenetic protein (BMP)-7 dramatically down-regulated expression of fibroblast markers, and that injection of BMP-7 adenoviral vectors into rats with advanced thioacetamide-induced cirrhosis abolished liver fibrosis.(18) BMP-7 is a potent inhibitor of EMT, and epithelial cells that are capable of transitioning to mesenchymal cells typically express BMP-7 to repress mesenchymal gene expression and maintain expression of E-cadherin and other epithelial genes.(19) Overexpressing BMP-7 in hepatocytes was shown to have no effect on hepatocyte E-cadherin expression, suggesting that the beneficial effects of BMP-7 on liver fibrosis were not due to inhibition of hepatocyte EMT.(18) However, the possibility that BMP-7 might be inhibiting EMT in some other type of liver cell was not examined. Previously, we proved that Shh induces ductular-type progenitor cells to undergo EMT.(20) We also showed that freshly-isolated Q-HSC express biliary epithelial markers, including E-cadherin, and the Hh antagonist, Hhip, and demonstrated that Q-HSC down-regulate Hhip, activate Hh signaling, lose epithelial markers, and express mesenchymal genes as they become MF-HSC. Blocking Hh signaling by pharmacologic inhibition repressed mesenchymal gene expression, while restoring expression of epithelial genes and other Q-HSC markers.(21) In the current study, we show that manipulating Rac1 in rat primary HSC, clonally-derived rat and human MF-HSC lines, and two different animal models of liver fibrosis consistently modulates Hh signaling, EMT, accumulation of MF-HSC, and fibrogenesis. These findings prove that Rac1 and Hh signaling are crucial mediators of HSC activation, confirm our hypothesis that these factors interact to induce and maintain the myofibroblastic HSC phenotype, identify key Hh pathway regulators as targets of activated Rac1, and suggest a novel mechanism for liver fibrosis. The latter involves activated Rac1-dependent induction of Shh and concomitant repression of Hhip, resultant activation of Hh signaling, and consequent transition of Q-HSC to MF-HSC, as well as enhanced MF-HSC viability. Together, these events cause accumulation of MF-HSC and increase fibrogenesis.