MTOR Overactivation In The Mesenchymal Cell Aggravates CCl₄-Induced Liver Fibrosis

BackgroundLiver fibrosis is a major cause of morbidity and mortality worldwide due to chronic viral hepatitis, alcoholic hepatitis and nonalcoholic steatohepatitis. Following liver injury, fibrosis as a wound-healing response is characterized by the accumulation of extracellular matrix (ECM). In recent years, advances in understanding the pathophysiology and mechanisms of hepatic fibrogenesis have been made. Myofibroblasts(MF) are defined primarily by their ability to produce ECM and exhibit contractile activity. Hepatic stellate cells (HSCs) are the major source of myofibroblasts in CCl4-induced mouse model. The paradigm in liver injury of activation of quiescent vitamin A-rich stellate cells into proliferative, contractile, and fibrogenic myofibroblasts has launched an era of astonishing progress in understanding the mechanistic basis of hepatic fibrosis progression and regression. But this simple paradigm has now yielded to a remarkably broad appreciation of the cell’s functions not only in liver injury, but also in hepatic development, regeneration, xenobiotic responses, intermediary metabolism, and immunoregulation. At the same time, countless studies have used tissue immunohistochemistry to identify stellate cells in liver, but the most prominent proteins analyzed to date include α-SMA, desmin, and GFAP.The mammalian target of rapamycin (mTOR) nucleates two distinct multi-protein complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). mTORC1 has five components including mTOR, Raptor,mLST8, PRAS40 and Deptor; mTORC2 has six components including mTOR,Rictor,mSIN1,Protor-1,LST8 and Deptor. The tuberous sclerosis complex (TSC), which comprises TSC1 and TSC2, involved in the negative regulation of mTORC1 activity. Loss of TSC1 causes cells and tissues to display constitutive mTORC1 activation. mTORC1 uniquely contains five components, in response to numerous intracellular and extracellular stimuli, mTORC1 phosphorylates eukaryotic initiation factor 4E-binding protein-1 (4E-BP1) and S6 kinase 1 (S6K1), exerts an essential role in increasing translation of a subset of mRNAs and accelerating growth and proliferation. Under certain circumstances, the inhibition of mTOR activity by rapamycin accelerates apoptosis. Activating mutations of mTOR can contribute to apoptotic resistance and might contribute to cellular transformation.mTOR pathway activation involved in fibrotic diseases has been reported. The mTOR pathway plays an important role in cardiac fibrosis, rapamycin is a potential therapeutic treatment that can be used to attenuate cardiac fibrosis. The rapamycin analogue SDZ RAD has dramatic inhibitory effects on lung-collagen accumulation in the bleomycin model of pulmonary fibrosis, suppression of mTOR may be used for treatment of pulmonary fibrosis. mTORC1 signaling promotes the activation of kidney fibroblasts and contributes to the development of interstitial fibrosis. There is growing evidence to support the idea that the mTOR signaling pathway plays a key role in liver fibrosis. HSCs activation are regarded as critical steps in the pathogenesis of liver fibrosis. HSCs are a resident mesenchymal cell type located in the subendothelial space of Disse, interposed between sinusoidal endothelium and hepatocytes. Based on these studies, we speculate that the activation of mTOR signaling in mesenchymal cell might also play a role in liver fibrosis.To date, the specific effect of mTOR on the liver fibrosis has not yet been reported. In this study, we aim to investigate the function of mTOR overactivation in mesenchymal cell in CCl4-induced liver fibrosis and to reveal the possible mechanism for for its participation in this disease.Methods1. Using Cre-loxp system. we have successfully created mice with mesenchymal cell specific deletion of TSC1.The genotype of the mice was identified by PCR and agarose gel electrophoresis for mice.2. After tamoxifen treatment, the effect of deletion determined by western blot and immunofluorescence for protein expression in liver tissues.3. Intraperitoneal injection of CC14 imitate the models of hepatic fibrosis. The control group received the same dose of olive oil. The severity of liver fibrosis is estimated according by serum ALT, tissue homogenates hydroxyproline (HYP), HE and Sirius red staining on tissue sections.4. Liver tissue samples from oil or CC14 treated WT or CKO mice at the 8 weeks were analyzed for a-SMA protein by Western blot analysis and Immunohistochemistry.5. To quantitate the expression of profibrogenic markers, RT-PCR was performed.6. To further ensure the importance of mTOR activity in myofibroblasts proliferation and apoptosis in CCl4-induced liver fibrosis, protein expression was monitored by western blot and immunofluorescence.Statistical analysisAll of the data were expressed as the mean ±SEM from three individual experiments. The data in each group were analyzed using t test (SPSS). Densitometry was performed using Image-Pro Plus. P values< 0.05 were considered statistically significant.Result1. Generation of TSC1-conditional knockout mice in the Mesenchymal CompartmentHomozygous floxed TSC1 mice (TSC1fl/fl) were cross bred with mice harboring a Cre-ER(T) recombinase gene to generate Cre/TSC1 heterozygous mice. The second cross generated [TSC1fll/fl, ColIa2-CreER(T)+/0] mice. Genomic DNA from tail tissue samples of these mice was genotyped by polymerase chain reaction (PCR) analysis. The presence of the TSC1 and Cre genes was confirmed (Figure 1A). To delete the TSC1 gene in the mesenchymal compartments, mice with genotype [TSC1fl/fl, ColIa2-CreER(T)+/0] were treated with tamoxifen as described in Materials and Methods and were referred to as TSC1 CKO mice. Mice with genotype [TSC1+1+, Colla2-CreER(T)+/0; TSC1flfl, ColIa2-CreER(T)0/0] under the same treatment were referred to as WT mice. The mesenchymal compartment deficiency of TSC1 expression was verified using Western blot analysis and immunofluorescence in liver WT and TSC1 CKO mice.2. TSC1 deletion augments CCL4-induced liver fibrosis in mice.To evaluate the effect of TSC1 deficiency in mesenchymal cell on liver fibrosis in vivo, TSC1 CKO and WT mice were treated with CCl4, and the fibrotic response was evaluated. A histologic examination of liver sections using H&E and Sirius red staining was performed. In TSC1 CKO mice exhibited worsen lobular architecture damage and severe and progressive bridging of necrosis than WT mice.TSC1 CKO mice presented exacerbated fibrosis by the morphometric assessment of the total area with positive Sirius Red staining and by higher liver hydroxyproline content. In comparison, TSC1 CKO mice exhibited higher serum ALT relative to WT mice. Collectively, these results demonstrated that TSC1 deletion in the mesenchymal Compartment augments CCl4-induced liver fibrosis in mice.3. Increased a-SMA expression due to loss of TSC1.Given that our previous analyses had determined that loss of TSC1 resulted in exacerbation in CCl4-induced hepatic fibrosis. Consistent with the visualized by increased liver fibrosis and collagen production, increased a-SMA positive staining were present in TSC1 CKO animals, in the presence of CCl4. No apparent difference in the expression of a-SMA was detected after oil treatment in both WT and CKO mice. Western blot analysis confirmed significant CCl4-induced increases in a-SMA protein in TSC1 CKO mice, the protein samples prepared from oil and CCl4-treated animals. These results suggest that TSC1 deletion from mesenchymal cell promote the development of liver fibrosis.4. Increased expression of profibrogenic markers in TSC1fll/fl mice.To quantitate the expression of profibrogenic markers, RT-PCR was performed. Levels of these markers were unchanged between the two genotypes from treatment with oil. TSC1 CKO mice exhibited increased expression of a-SMA, collagen I, TGF-P and TIMP2 in CCl4-induced animals relative to WT mice. This conclusion strengthen the evidence for the vulnerability to CCl4-induced fibrosis in TSC1 CKO mice.5. Increased myofibroblasts in TSC1 CKO Mice.To further ensure the importance of mTOR activity in myofibroblasts proliferation in CCl4-induced liver fibrosis, protein expression was monitored by western blot and immunofluorescence. As expected, pS6 expression was upregulated in those from CCl4-treated mice, but was the highest in the TSC1 CKO mouse. We further detected the protein level of apoptotic proteins, the cleaved form of caspase-3 and PARP reduced in CCl4-treated mice, but was lowest in the TSC1 CKO mouse. Myofibroblast formation as measured by immunofluorescence analysis with a-SMA antibody. Consistent with the WB results, in CCL4 group increased expression of pS6 at least partially caused the elevated myofibroblast formation seen in TSC1 CKO animals. In CCL4 group, ki67 and a-SMA double-positive cells, which are recognized as proliferative myofibroblasts, were significantly increased to a greater extent in TSC1 CKO mice compared with WT mice. Cleaved caspas3 and a-SMA double-positive cells, which are recognized as apoptotic myofibroblasts were decreased in TSC1 CKO mice compared with WT mice. Intriguingly, a-SMA, ki67 and cleaved caspas3 expression were not appreciably altered in oil group mice, indicating that in quiescent condition loss of TSC1 in collagen type I expressing cells was not accompanied by the increased appearance of myofibroblasts in liver.Thus, TSC1 deficiency in the mesenchymal compartment resulted in significantly increased myofibroblast contributing to the noted aggravation in liver fibrosis.ConclusionIn summary, mTOR overactivation in the mesenchymal compartment augmented liver fibrosis induced by CCl4. The mechanism likely involved that activation of mTOR induces myofibroblast proliferation and prevents apoptosis.