Tissue and extracellular matrix (ECM) stiffness is transduced into intracellular stiffness, signaling, and changes in cellular behavior. Integrins and several of their associated focal adhesion proteins have been implicated in sensing ECM stiffness. We investigated how an initial sensing event is translated into intracellular stiffness and a biologically interpretable signal. We found that a pathway consisting of focal adhesion kinase (FAK), the adaptor protein p130Cas (Cas), and the guanosine triphosphatase Rac selectively transduced ECM stiffness into stable intracellular stiffness, increased abundance of the cell cycle protein cyclin D1, and promoted S phase entry. Rac-dependent intracellular stiffening involved its binding partner lamellipodin, a protein that transmits Rac signals to the cytoskeleton during cell migration. Our findings establish that mechanotransduction by a FAK-Cas-Rac-lamellipodin signaling module converts the external information encoded by ECM stiffness into stable intracellular stiffness and mechanosensitive cell cycling. Thus, lamellipodin is not only important in controlling cellular migration, but also for regulating the cell cycle in response to mechanical signals.

Transdifferentiation is a complete and stable change in cell identity that serves as an alternative to stem-cell-mediated organ regeneration. In adult mammals, findings of transdifferentiation have been limited to the replenishment of cells lost from preexisting structures, in the presence of a fully developed scaffold and niche. Here we show that transdifferentiation of hepatocytes in the mouse liver can build a structure that failed to form in development-the biliary system in a mouse model that mimics the hepatic phenotype of human Alagille syndrome (ALGS). In these mice, hepatocytes convert into mature cholangiocytes and form bile ducts that are effective in draining bile and persist after the cholestatic liver injury is reversed, consistent with transdifferentiation. These findings redefine hepatocyte plasticity, which appeared to be limited to metaplasia, that is, incomplete and transient biliary differentiation as an adaptation to cell injury, based on previous studies in mice with a fully developed biliary system. In contrast to bile duct development, we show that de novo bile duct formation by hepatocyte transdifferentiation is independent of NOTCH signalling. We identify TGFβ signalling as the driver of this compensatory mechanism and show that it is active in some patients with ALGS. Furthermore, we show that TGFβ signalling can be targeted to enhance the formation of the biliary system from hepatocytes, and that the transdifferentiation-inducing signals and remodelling capacity of the bile-duct-deficient liver can be harnessed with transplanted hepatocytes. Our results define the regenerative potential of mammalian transdifferentiation and reveal opportunities for the treatment of ALGS and other cholestatic liver diseases.

Highlights d Random lineage tracing provides a representative sample of all hepatocytes d Liver homeostasis relies on modest proliferation of hepatocytes in all zones d The burden of proliferation in liver regeneration is distributed among hepatocytes d Chronic injury reveals differences in hepatocyte proliferation caused by ploidy

SUMMARY Liver fibrosis, a form of scarring, gradually develops in chronic liver diseases when hepatocyte regeneration cannot compensate for hepatocyte death. At earlier stages, collagen produced by activated myofibroblasts (MFs) functions to maintain tissue integrity, but upon repeated injury, collagen accumulation suppresses hepatocyte regeneration, ultimately leading to liver failure. As a strategy to generate new hepatocytes and limit collagen deposition in the chronically injured liver, we developed in vivo reprogramming of MFs into hepatocytes using adeno-associated virus (AAV) vectors expressing hepatic transcription factors. We first identified the AAV6 subtype as effective in transducing MFs in mouse models of chronic liver disease. We then use lineage-tracing approaches to show that hepatocytes reprogrammed from MFs replicate primary hepatocyte function, and that liver fibrosis in AAV treated animals is reduced. Because AAV vectors are already used for liver-directed human gene therapy, our strategy has potential for clinical translation into a therapy for liver fibrosis.

Apolipoprotein E3 (apoE3) is thought to protect against atherosclerosis by enhancing reverse cholesterol transport. However, apoE3 also has cholesterol-independent effects that contribute to its anti-atherogenic properties. These include altering extracellular matrix protein synthesis and inhibiting vascular smooth muscle cell proliferation. Both of these cholesterol-independent effects result from an apoE3-mediated induction of cyclooxygenase-2 (Cox2). Nevertheless, how apoE3 regulates Cox2 remains unknown. Here, we show that apoE3 inhibits the activation of Rho, which reduces the formation of actin stress fibers and focal adhesions and results in cellular softening. Inhibition of Rho-Rho kinase signaling or direct cellular softening recapitulates the effect of apoE3 on Cox2 expression while a constitutively active Rho mutant overrides the apoE3 effect on both intracellular stiffness and Cox2. Thus, our results describe a previously unidentified mechanism by which an atheroprotective apolipoprotein uses Rho to control cellular mechanics and Cox2.

Background and Aims Patient‐derived human‐induced pluripotent stem cells (hiPSCs) differentiated into hepatocytes (hiPSC‐Heps) have facilitated the study of rare genetic liver diseases. Here, we aimed to establish an in vitro liver disease model of the urea cycle disorder ornithine transcarbamylase deficiency (OTCD) using patient‐derived hiPSC‐Heps. Approach and Results Before modeling OTCD, we addressed the question of why hiPSC‐Heps generally secrete less urea than adult primary human hepatocytes (PHHs). Because hiPSC‐Heps are not completely differentiated and maintain some characteristics of fetal PHHs, we compared gene‐expression levels in human fetal and adult liver tissue to identify genes responsible for reduced urea secretion in hiPSC‐Heps. We found lack of aquaporin 9 (AQP9) expression in fetal liver tissue as well as in hiPSC‐Heps, and showed that forced expression of AQP9 in hiPSC‐Heps restores urea secretion and normalizes the response to ammonia challenge by increasing ureagenesis. Furthermore, we proved functional ureagenesis by challenging AQP9‐expressing hiPSC‐Heps with ammonium chloride labeled with the stable isotope [15N] (15NH4Cl) and by assessing enrichment of [15N]‐labeled urea. Finally, using hiPSC‐Heps derived from patients with OTCD, we generated a liver disease model that recapitulates the hepatic manifestation of the human disease. Restoring OTC expression—together with AQP9—was effective in fully correcting OTC activity and normalizing ureagenesis as assessed by 15NH4Cl stable‐isotope challenge. Conclusion Our results identify a critical role for AQP9 in functional urea metabolism and establish the feasibility of in vitro modeling of OTCD with hiPSC‐Heps. By facilitating studies of OTCD genotype/phenotype correlation and drug screens, our model has potential for improving the therapy of OTCD.