PDGF receptor-? promotes TGF-? signaling in hepatic stellate cells via transcriptional and posttranscriptional regulation of TGF-? receptors.
ABSTRACT: Platelet-derived growth factor (PDGF) and transforming growth factor-? (TGF-?) signaling are required for hepatic stellate cell (HSC) activation under pathological conditions such as liver metastatic tumor growth. These two signaling pathways are functionally divergent; PDGF signaling promotes proliferation and migration of HSCs, and TGF-? induces transdifferentiation of quiescent HSCs into myofibroblasts. Although PDGF signaling is implicated in TGF-?-mediated epithelial mesenchymal transition of tumor cells, the role of PDGF receptors in TGF-? activation of HSCs has not been investigated. Here we report that PDGF receptor-? (PDGFR-?) is required for TGF-? signaling of cultured human HSCs although HSCs express both PDGF-? and -? receptors. PDGFR-? knockdown inhibits TGF-?-induced phosphorylation and nuclear accumulation of SMAD2 with no influence on AKT or ERK phosphorylation associated with noncanonical TGF-? signaling. PDGFR-? knockdown suppresses TGF-? receptor I (T?RI) but increases T?RII gene transcription. At the protein level, PDGFR-? is recruited to T?RI/T?RII complexes by TGF-? stimulation. PDGFR-? knockdown blocks TGF-?-mediated internalization of T?RII and induces accumulation of T?RII at the plasma membrane, thereby inhibiting TGF-? phosphorylation of SMAD2. Functionally, knockdown of PDGFR-? reduces paracrine effects of HSCs on colorectal cancer cell proliferation and migration in vitro. In mice and patients, colorectal cancer cell invasion of the liver induces upregulation of PDGFR-? of HSCs. In summary, our finding that PDGFR-? knockdown inhibits SMAD-dependent TGF-? signaling by repressing T?RI transcriptionally and blocking endocytosis of TGF-? receptors highlights a convergence of PDGF and TGF-? signaling for HSC activation and PDGFR-? as a therapeutic target for liver metastasis and other settings of HSC activation.
Project description:Glycyrrhetinic acid (GA), a hydrolysate of glycyrrhizic acid from licorice root extract, has been used to treat liver fibrotic diseases. However, the molecular mechanism involved in the antifibrotic effects of GA remains unclear. The involvement of miR-663a and its roles in TGF-?-1-induced hepatic stellate cell (HSC) activation remains unclear. In this study, we investigated the roles of miR-663a in the activation of HSCs and the antifibrosis mechanism of GA. MiR-663a expression was downregulated in TGF-?-treated HSCs. The overexpression of miR-663a inhibited HSC proliferation. TGF-?-1was confirmed as a direct target gene of miR-663a. MiR-663a alleviated HSC activation, concomitant with decreased expression of ?-smooth muscle actin (?-SMA), human ?2 (I) collagen (COL1A2), TGF-?1, TGF-?RI, Smad4, p-Smad2, and p-Smad3. GA upregulated miR-663a expression and inhibited the TGF-?/Smad pathway in HSCs. Further studies showed that miR-663a inhibitor treatment reversed GA-mediated downregulation of TGF-?1, TGF-?RI, Smad4, p-Smad2, p-Smad3, ?-SMA, and CoL1A2 in TGF-?1-treated HSCs. These results show that miR-663a suppresses HSC proliferation and activation and the TGF-?/Smad signaling pathway, highlighting that miR-663a can be utilized as a therapeutic target for hepatic fibrosis. GA inhibits, at least in part, HSC proliferation and activation via targeting the miR-663a/TGF-?/Smad signaling pathway.
Project description:Activation of hepatic stellate cells (HSCs) is a key event in the initiation of liver fibrosis, characterized by enhanced extracellular matrix production and altered degradation. Activation of HSCs can be modulated by cytokines produced by immune cells. Recent reports have implicated the proinflammatory cytokine IL-17A in liver fibrosis progression. We hypothesized that IL-17A may enhance activation of HSCs and induction of the fibrogenic signals in these cells. The human HSC line LX2 and primary human HSCs were stimulated with increasing doses of IL-17A and compared with TGF-?- and PBS-treated cells as positive and negative controls, respectively. IL-17A alone did not induce activation of HSCs. However, IL-17A sensitized HSCs to the action of suboptimal doses of TGF-? as confirmed by strong induction of ?-smooth muscle actin, collagen type I (COL1A1), and tissue inhibitor of matrix metalloproteinase I gene expression and protein production. IL-17A specifically upregulated the cell surface expression of TGF-?RII following stimulation. Pretreatment of HSCs with IL-17A enhanced signaling through TGF-?RII as observed by increased phosphorylation of SMAD2/3 in response to stimulation with suboptimal doses of TGF-?. This enhanced TGF-? response of HSCs induced by IL-17A was JNK-dependent. Our results suggest a novel profibrotic function for IL-17A by enhancing the response of HSCs to TGF-? through activation of the JNK pathway. IL-17A acts through upregulation and stabilization of TGF-?RII, leading to increased SMAD2/3 signaling. These findings represent a novel example of cooperative signaling between an immune cytokine and a fibrogenic receptor.
Project description:TGF? induces the differentiation of hepatic stellate cells (HSCs) into tumor-promoting myofibroblasts but underlying mechanisms remain incompletely understood. Because endocytosis of TGF? receptor II (T?RII), in response to TGF? stimulation, is a prerequisite for TGF signaling, we investigated the role of protein diaphanous homolog 1 (known as Diaph1 or mDia1) for the myofibroblastic activation of HSCs. Using shRNA to knockdown Diaph1 or SMIFH2 to target Diaph1 activity of HSCs, we found that the inactivation of Diaph1 blocked internalization and intracellular trafficking of T?RII and reduced SMAD3 phosphorylation induced by TGF?1. Mechanistic studies revealed that the N-terminal portion of Diaph1 interacted with both T?RII and Rab5a directly and that Rab5a activity of HSCs was increased by Diaph1 overexpression and decreased by Diaph1 knockdown. Additionally, expression of Rab5aQ79L (active Rab5a mutant) increased whereas the expression of Rab5aS34N (inactive mutant) reduced the endosomal localization of T?RII in HSCs compared to the expression of wild-type Rab5a. Functionally, TGF? stimulation promoted HSCs to express tumor-promoting factors, and ?-smooth muscle actin, fibronection, and CTGF, markers of myofibroblastic activation of HSCs. Targeting Diaph1 or Rab5a suppressed HSC activation and limited tumor growth in a tumor implantation mouse model. Thus, Dipah1 and Rab5a represent targets for inhibiting HSC activation and the hepatic tumor microenvironment.
Project description:Members of the transforming growth factor ? (TGF?) family initiate cellular responses by binding to TGF? receptor type II (T?RII) and type I (T?RI) serine/threonine kinases, whereby Smad2 and Smad3 are phosphorylated and activated, promoting their association with Smad4. We report here that T?RI interacts with the SH3 domains of the adaptor protein CIN85 in response to TGF? stimulation in a TRAF6-dependent manner. Small interfering RNA-mediated knockdown of CIN85 resulted in accumulation of T?RI in intracellular compartments and diminished TGF?-stimulated Smad2 phosphorylation. Overexpression of CIN85 instead increased the amount of T?RI at the cell surface. This effect was inhibited by a dominant-negative mutant of Rab11, suggesting that CIN85 promoted recycling of TGF? receptors. CIN85 enhanced TGF?-stimulated Smad2 phosphorylation, transcriptional responses, and cell migration. CIN85 expression correlated with the degree of malignancy of prostate cancers. Collectively, our results reveal that CIN85 promotes recycling of TGF? receptors and thereby positively regulates TGF? signaling.
Project description:Liver microenvironment is a critical determinant for development and progression of liver metastasis. Under transforming growth factor beta (TGF-?) stimulation, hepatic stellate cells (HSCs), which are liver-specific pericytes, transdifferentiate into tumor-associated myofibroblasts that promote tumor implantation (TI) and growth in the liver. However, the regulation of this HSC activation process remains poorly understood. In this study, we tested whether vasodilator-stimulated phosphoprotein (VASP) of HSCs regulated the TGF-?-mediated HSC activation process and tumor growth. In both an experimental liver metastasis mouse model and cancer patients, colorectal cancer cells reaching liver sinusoids induced up-regulation of VASP and alpha-smooth muscle actin (?-SMA) in adjacent HSCs. VASP knockdown in HSCs inhibited TGF-?-mediated myofibroblastic activation of HSCs, TI, and growth in mice. Mechanistically, VASP formed protein complexes with TGF-? receptor II (T?RII) and Rab11, a Ras-like small GTPase and key regulator of recycling endosomes. VASP knockdown impaired Rab11 activity and Rab11-dependent targeting of T?RII to the plasma membrane, thereby desensitizing HSCs to TGF-?1 stimulation.Our study demonstrates a requirement of VASP for TGF-?-mediated HSC activation in the tumor microenvironment by regulating Rab11-dependent recycling of T?RII to the plasma membrane. VASP and its effector, Rab11, in the tumor microenvironment thus present therapeutic targets for reducing TI and metastatic growth in the liver.
Project description:Transforming growth factor-? (TGF-?) receptors (T?Rs) are essential components for TGF-? signal transduction in T cells, yet the mechanisms by which the receptors are regulated remain poorly understood. We show here that Poly(ADP-ribose) polymerase-1 (PARP-1) regulates TGF-? receptor I (T?RI) and II (T?RII) expression in CD4(+) T cells and subsequently affects Smad2/3-mediated TGF-? signal transduction. Inhibition of PARP-1 led to the upregulation of both T?RI and T?RII, yet the underlying molecular mechanisms were distinct. PARP-1 selectively bound to the promoter of T?RII, whereas the enzymatic activity of PARP-1 was responsible for the inhibition of T?RI expression. Importantly, inhibition of PARP-1 also enhanced expression of T?Rs in human CD4(+) T cells. Thus, PARP-1 regulates T?R expression and TGF-? signaling in T cells.
Project description:Liver fibrosis is characterized by the activation and migration of hepatic stellate cells (HSCs), followed by matrix deposition. Recently, several studies have shown the importance of extracellular vesicles (EVs) derived from liver cells, such as hepatocytes and endothelial cells, in liver pathobiology. While most of the studies describe how liver cells modulate HSC behavior, an important gap exists in the understanding of HSC-derived signals and more specifically HSC-derived EVs in liver fibrosis. Here, we investigated the molecules released through HSC-derived EVs, the mechanism of their release, and the role of these EVs in fibrosis. Mass spectrometric analysis showed that platelet-derived growth factor (PDGF) receptor-alpha (PDGFR?) was enriched in EVs derived from PDGF-BB-treated HSCs. Moreover, patients with liver fibrosis had increased PDGFR? levels in serum EVs compared to healthy individuals. Mechanistically, in vitro tyrosine720-to-phenylalanine mutation on the PDGFR? sequence abolished enrichment of PDGFR? in EVs and redirected the receptor toward degradation. Congruently, the inhibition of Src homology 2 domain tyrosine phosphatase 2, the regulatory binding partner of phosphorylated tyrosine720, also inhibited PDGFR? enrichment in EVs. EVs derived from PDGFR?-overexpressing cells promoted in vitro HSC migration and in vivo liver fibrosis. Finally, administration of Src homology 2 domain tyrosine phosphatase 2inhibitor, SHP099, to carbon tetrachloride-administered mice inhibited PDGFR? enrichment in serum EVs and reduced liver fibrosis. CONCLUSION:PDGFR? is enriched in EVs derived from PDGF-BB-treated HSCs in an Src homology 2 domain tyrosine phosphatase 2-dependent manner and these PDGFR?-enriched EVs participate in development of liver fibrosis. (Hepatology 2018;68:333-348).
Project description:Hepatic stellate cell (HSC) activation is a pivotal event in initiation and progression of hepatic fibrosis and a major contributor to collagen deposition driven by transforming growth factor beta (TGF-?). MicroRNAs (miRs), small noncoding RNAs modulating messenger RNA (mRNA) and protein expression, have emerged as key regulatory molecules in chronic liver disease. We investigated differentially expressed miRs in quiescent and activated HSCs to identify novel regulators of profibrotic TGF-? signaling. miR microarray analysis was performed on quiescent and activated rat HSCs. Members of the miR-17-92 cluster (19a, 19b, 92a) were significantly down-regulated in activated HSCs. Because miR 19b showed the highest fold-change of the cluster members, activated HSCs were transfected with miR 19b mimic or negative control and TGF-? signaling and HSC activation assessed. miR 19b expression was determined in fibrotic rat and human liver specimens. miR 19b mimic negatively regulated TGF-? signaling components demonstrated by decreased TGF-? receptor II (TGF-?RII) and SMAD3 expression. Computational prediction of miR 19b binding to the 3' untranslated region of TGF-?RII was validated by luciferase reporter assay. Inhibition of TGF-? signaling by miR 19b was confirmed by decreased expression of type I collagen and by blocking TGF-?-induced expression of ?1(I) and ?2(I) procollagen mRNAs. miR 19b blunted the activated HSC phenotype by morphological assessment and decreased smooth muscle ?-actin expression. Additionally, miR 19b expression was markedly diminished in fibrotic rat liver compared with normal liver; similarly, miR 19b expression was markedly down-regulated in fibrotic compared with normal human livers.miR 19b is a novel regulator of TGF-? signaling in HSCs, suggesting a potential therapeutic approach for hepatic fibrosis.
Project description:Transforming growth factor ? (TGF?) potently activates hepatic stellate cells (HSCs), which promotes production and secretion of extracellular matrix (ECM) proteins and hepatic fibrogenesis. Increased ECM synthesis and secretion in response to TGF? is associated with endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). TGF? and UPR signaling pathways are tightly intertwined during HSC activation, but the regulatory mechanism that connects these two pathways is poorly understood. Here, we found that TGF? treatment of immortalized HSCs (i.e. LX-2 cells) induces phosphorylation of the UPR sensor inositol-requiring enzyme 1? (IRE1?) in a SMAD2/3-procollagen I-dependent manner. We further show that IRE1? mediates HSC activation downstream of TGF? and that its role depends on activation of a signaling cascade involving apoptosis signaling kinase 1 (ASK1) and c-Jun N-terminal kinase (JNK). ASK1-JNK signaling promoted phosphorylation of the UPR-associated transcription factor CCAAT/enhancer binding protein ? (C/EBP?), which is crucial for TGF?- or IRE1?-mediated LX-2 activation. Pharmacological inhibition of C/EBP? expression with the antiviral drug adefovir dipivoxil attenuated TGF?-mediated activation of LX-2 or primary rat HSCs in vitro and hepatic fibrogenesis in vivo Finally, we identified a critical relationship between C/EBP? and the transcriptional regulator p300 during HSC activation. p300 knockdown disrupted TGF?- or UPR-induced HSC activation, and pharmacological inhibition of the C/EBP?-p300 complex decreased TGF?-induced HSC activation. These results indicate that TGF?-induced IRE1? signaling is critical for HSC activation through a C/EBP?-p300-dependent mechanism and suggest C/EBP? as a druggable target for managing fibrosis.
Project description:When skin is wounded, migration of epidermal keratinocytes at the wound edge initiates within hours, whereas migration of dermal fibroblasts toward the wounded area remains undetectable until several days later. This "cell type traffic" regulation ensures proper healing of the wound, as disruptions of the regulation could either cause delay of wound healing or result in hypertrophic scars. TGF?3 is the critical traffic controller that selectively halts migration of the dermal, but not epidermal, cells to ensure completion of wound re-epithelialization prior to wound remodeling. However, the mechanism of TGF?3's anti-motility signaling has never been investigated. We report here that activated T?RII transmits the anti-motility signal of TGF?3 in full to T?RI, since expression of the constitutively activated T?RI-TD mutant was sufficient to replace TGF?3 to block PDGF-bb-induced dermal fibroblast migration. Second, the three components of R-Smad complex are all required. Individual downregulation of Smad2, Smad3 or Smad4 prevented TGF?3 from inhibiting dermal fibroblast migration. Third, Protein Kinase Array allowed us to identify the protein kinase A (PKA) as a specific downstream effector of R-Smads in dermal fibroblasts. Activation of PKA alone blocked PDGF-bb-induced dermal fibroblast migration, just like TGF?3. Downregulation of PKA's catalytic subunit nullified the anti-motility signaling of TGF?3. This is the first report on anti-motility signaling mechanism by TGF? family cytokines. Significance of this finding is not only limited to wound healing but also to other human disorders, such as heart attack and cancer, where the diseased cells have often managed to avoid the anti-motility effect of TGF?.