TGF-β1 induces endothelial cell apoptosis by shifting VEGF activation of p38(MAPK) from the prosurvival p38β to proapoptotic p38α

TGF-β1 and VEGF, both angiogenesis inducers, have opposing effects on vascular endothelial cells. TGF-β1 induces apoptosis; VEGF induces survival. We have previously shown that TGF-β1 induces endothelial cell expression of VEGF, which mediates TGF-β1 induction of apoptosis through activation of p38 mitogen-activated protein kinase (MAPK). Because VEGF activates p38(MAPK) but protects the cells from apoptosis, this finding suggested that TGF-β1 converts p38(MAPK) signaling from prosurvival to proapoptotic. Four isoforms of p38(MAPK) -α, β, γ, and δ-have been identified. Therefore, we hypothesized that different p38(MAPK) isoforms control endothelial cell apoptosis or survival, and that TGF-β1 directs VEGF activation of p38(MAPK) from a prosurvival to a proapoptotic isoform. Here, we report that cultured endothelial cells express p38α, β, and γ. VEGF activates p38β, whereas TGF-β1 activates p38α. TGF-β1 treatment rapidly induces p38α activation and apoptosis. Subsequently, p38α activation is downregulated, p38β is activated, and the surviving cells become refractory to TGF-β1 induction of apoptosis and proliferate. Gene silencing of p38α blocks TGF-β1 induction of apoptosis, whereas downregulation of p38β or p38γ expression results in massive apoptosis. Thus, in endothelial cells p38α mediates apoptotic signaling, whereas p38β and p38γ transduce survival signaling. TGF-β1 activation of p38α is mediated by VEGF, which in the absence of TGF-β1 activates p38β. Therefore, these results show that TGF-β1 induces endothelial cell apoptosis by shifting VEGF signaling from the prosurvival p38β to the proapoptotic p38α.     

Canonical Wnt signaling skews TGF-β signaling in chondrocytes towards signaling via ALK1 and Smad 1/5/8

BackgroundBoth Wnt signaling and TGF-β signaling have been implicated in the regulation of the phenotype of many cell types including chondrocytes, the only cell type present in the articular cartilage. A changed chondrocyte phenotype, resulting in chondrocyte hypertrophy, is one of the main hallmarks of osteoarthritis. TGF-β signaling via activin-like kinase (ALK)5, resulting in Smad 2/3 phosphorylation, inhibits chondrocyte hypertrophy. In contrast, TGF-β signaling via ALK1, leading to Smad 1/5/8 phosphorylation, has been shown to induce chondrocyte hypertrophy. In this study, we investigated the capability of Wnt3a and WISP1, a protein downstream in canonical Wnt signaling, to skew TGF-β signaling in chondrocytes from the protective Smad 2/3 towards the Smad 1/5/8 pathway.ResultsStimulation with Wnt3a, either alone or in combination with its downstream protein WISP1, decreased TGF-β-induced C-terminal phosphorylation of Smad 2/3. In addition, both Wnt3a and WISP1 increased Smad 1/5/8 phosphorylation at the C-terminal domain in both murine and human chondrocytes. DKK-1, a selective inhibitor of canonical Wnt signaling, abolished these effects. TGF-β signaling via Smad 2/3, measured by the functional CAGA₁₂-Luc reporter construct activity, was decreased by stimulation with Wnt3a in accordance with the decrease in Smad 2/3 phosphorylation found on Western blot. Furthermore, in vivo overexpression of the canonical Wnt8a decreased Smad 2/3 phosphorylation and increased Smad 1/5/8 phosphorylation.ConclusionsOur data show that canonical Wnt signaling is able to skew TGF-β signaling towards dominant signaling via the ALK1/Smad 1/5/8 pathway, which reportedly leads to chondrocyte hypertrophy. In this way canonical Wnts and WISP1, which we found to be increased during experimental osteoarthritis, may contribute to osteoarthritis pathology.     

Lipopolysaccharide inhibits transforming growth factor-beta1-stimulated Smad6 expression by inducing phosphorylation of the linker region of Smad3 through a TLR4-IRAK1-ERK1/2 pathway

Smad6, one of the inhibitory Smads, plays an important role in transforming growth factor-beta1 (TGF-β1)-mediated negative regulation of pro-inflammatory signaling. In this study, we found that bacterial endotoxin lipopolysaccharide (LPS) inhibits TGF-β1-induced expression of Smad6 in RAW264.7 cells. This repression was accompanied by increased Smad3 linker phosphorylation at Thr-179 and Ser-208 and was dependent on ERK1/2 activity via the TLR4-IRAK1-linked signaling cascade. The expression of a mutant Smad3, that lacks the phosphorylation sites in the linker regions, significantly reversed the inhibitory effect of LPS on TGF-β1-induced Smad6 expression and its anti-inflammatory capacity. Collectively, our findings show how LPS pro-inflammatory signal antagonizes the anti-inflammatory activity of TGF-β1.     

The role of Smad signaling in vascular and hematopoietic development revealed by studies using genetic mouse models

Smads are intracellular mediators of transforming growth factor β (TGF-β) superfamily signaling. In this review, we focus on the genetic mouse models for Smad pathways, which have provided functional evidence regarding the complex circuitry in angiogenesis and hematopoiesis during development. In the early stages of vascular development, TGF-β signaling is a contri buting factor in angiogenesis and vascular maturation. Whereas in the later embryogenesis, selected molecules of Smad pathways, such as TGF-β type II receptor (TbRII), ALK5, and Smad5, seem to be dispensable for vessel morphogenesis and integrity. TGF-β signaling is not required in the induction of hematopoietic precursors from mesoderm, but inhibits the subsequent expansion of committed hematopoietic precursors. By contrast, bone morphogenetic protein 4 (BMP4) has long been acknowledged pivotal in mesoderm induction and hematopoietic commitment during development. However, recent genetic evidence shows the BMP4-ALK3 axis is not crucial for the formation of hematopoietic cells from FLK1+ mesoderm. Because of the highly redundant mechanisms within the Smad pathways, the precise role of the Smad signaling involved in vascular and hematopoietic development remains nebulous. The generation of novel cell lineage restricted Cre transgenes would shed new light on the future relevant investigations.     

SARA is dispensable for functional TGF-β signaling

Smad anchor for receptor activation (SARA or ZFYVE9) has been proposed to mediate transforming growth factor β (TGF-β) signaling by direct interaction with the non-activated Smad proteins and the TGF-β receptors; however, these findings are controversial. We demonstrate no correlation between SARA expression and the levels of TGF-β-induced phosphorylation of Smads in various B-cell lymphomas. Moreover, knockdown of SARA in HeLa cells did not interfere with TGF-β-induced Smad activation, Smad nuclear translocation, or induction of TGF-β target genes. Various R-Smads and TGF-β receptors did not co-immunoprecipitate with SARA. Collectively, our results demonstrate that SARA is dispensable for functional TGF-β-mediated signaling.     

TGF-β1 exerts opposing effects on grass carp leukocytes: implication in teleost immunity, receptor signaling and potential self-regulatory mechanisms

In fish immunity, the regulatory role of transforming growth factor-β1 (TGF-β1) has not been fully characterized. Here we examined the immunoregulatory effects of TGF-β1 in grass carp peripheral blood leukocytes (PBL) and head kidney leukocytes (HKL). It is interesting that TGF-β1 consistently stimulated the cell viability and the mRNA levels of pro-inflammatory cytokines (Tnfα and Ifnγ) and T/B cell markers [Cd4-like (Cd4l), Cd8α, Cd8β and Igμ] in PBL, which contrasted with its inhibitory tone in HKL. Further studies showed that grass carp TGF-β1 type I receptor, activin receptor-like kinase 5 (ALK5), was indispensable for the immunoregulatory effects of TGF-β1 in PBL and HKL. Notably, TGF-β1 persistently attenuated ALK5 expression, whereas immunoneutralization of endogenous grass carp TGF-β1 could increase ALK5 mRNA and protein levels. It is consistent with the observation that TGF-β1 decreased the number of ALK5(+) leukocytes in PBL and HKL, revealing a negative regulation of TGF-β1 signaling at the receptor level. Moreover, transient treatment with TGF-β1 for 24 h was sufficient to induce similar cellular responses compared with the continuous treatment. This indicated a possible mechanism by which TGF-β1 triggered the down-regulation of ALK5 mRNA and protein, leading to the desensitization of grass carp leukocytes toward TGF-β1. Accordingly, our data revealed a dual role of TGF-β1 in teleost immunity in which it can serve as a positive or negative control device and provided additional mechanistic insights as to how TGF-β1 controls its signaling in vertebrate leukocytes.     

Transforming growth factor-β1 suppresses hepatitis B virus replication by the reduction of hepatocyte nuclear factor-4α expression

Several studies have demonstrated that cytokine-mediated noncytopathic suppression of hepatitis B virus (HBV) replication may provide an alternative therapeutic strategy for the treatment of chronic hepatitis B infection. In our previous study, we showed that transforming growth factor-beta1 (TGF-β1) could effectively suppress HBV replication at physiological concentrations. Here, we provide more evidence that TGF-β1 specifically diminishes HBV core promoter activity, which subsequently results in a reduction in the level of viral pregenomic RNA (pgRNA), core protein (HBc), nucleocapsid, and consequently suppresses HBV replication. The hepatocyte nuclear factor 4alpha (HNF-4α) binding element(s) within the HBV core promoter region was characterized to be responsive for the inhibitory effect of TGF-β1 on HBV regulation. Furthermore, we found that TGF-β1 treatment significantly repressed HNF-4α expression at both mRNA and protein levels. We demonstrated that RNAi-mediated depletion of HNF-4α was sufficient to reduce HBc synthesis as TGF-β1 did. Prevention of HNF-4α degradation by treating with proteasome inhibitor MG132 also prevented the inhibitory effect of TGF-β1. Finally, we confirmed that HBV replication could be rescued by ectopic expression of HNF-4α in TGF-β1-treated cells. Our data clarify the mechanism by which TGF-β1 suppresses HBV replication, primarily through modulating the expression of HNF-4α gene.     

Blocking core fucosylation of TGF-β1 receptors downregulates their functions and attenuates the epithelial-mesenchymal transition of renal tubular cells

Posttranslational modification of proteins could regulate their multiple biological functions. Transforming growth factor-β receptor I and II (ALK5 and TGF-βRII), which are glycoproteins, play important roles in the renal tubular epithelial-mesenchymal transition (EMT). In the present study, we examined the role of core fucosylation of TGF-βRII and ALK5, which is regulated by α-1,6 fucosyltransferase (Fut8), in the process of EMT of cultured human renal proximal tubular epithelial (HK-2) cells. The typical cell model of EMT induced by TGF-β1 was constructed to address the role of core fucosylation in EMT. Core fucosylation was found to be essential for both TGF-βRII and ALK5 to fulfill their functions, and blocking it with Fut8 small interfering RNA greatly reduced the phosphorylation of Smad2/3 protein, caused the inactivation of TGF-β/Smad2/3 signaling, and resulted in remission of EMT. More importantly, even with high levels of expressions of TGF-β1, TGF-βRII, and ALK5, blocking core fucosylation also could attenuate the EMT of HK-2 cells. Thus blocking core fucosylation of TGF-βRII and ALK5 may attenuate EMT independently of the expression of these proteins. This study may provide new insight into the role of glycosylation in renal interstitial fibrosis. Furthermore, core fucosylation may be a novel potential therapeutic target for treatment of renal tubular EMT.     

TNF-α suppressed TGF-β-induced CTGF expression by switching the binding preference of p300 from Smad4 to p65

TGF-β regulates diverse biologic effects including cell growth, cell death or apoptosis, cell differentiation, and extracellular matrix (ECM) synthesis. Connective tissue growth factor (CTGF), induced by TGF-β has been reported to mediate stimulatory action of TGF-β-induced ECM. Although TNF-α was reported to suppress the TGF-β-induced CTGF gene expression, the molecular mechanism is not well clarified. In this study, we found the inhibitory effect of TNF-α on TGF-β-induced CTGF expression in WT but not p65?/? MEF cells. TNF-α neither induced Smad7 expression nor affected TGF-β-induced Smad2 phosphorylation and nuclear translocation. We demonstrated that p300 physically associated with p65 rather than Smad4 in the presence of both TNF-α and TGF-β. Moreover, the TGF-β-induced binding of p300 and acetylated H4, but not Smad4 to the CTGF promoter was disturbed by TNF-α treatment. Overall, our data showed that suppression of TNF-α on TGF-β-induced CTGF expression is due to the competition of p300 by p65 and Smad4.