Linker Region Phosphorylation Research Articles

Cardioprotective effect of tamoxifen and raloxifene: Preventing proteoglycan synthesis by modulating non-canonical TGF-β signalling through NADPH oxidase and ERK phosphorylation

Atherosclerosis, a leading cause of cardiovascular disease, remains a significant global health concern. Tamoxifen and raloxifene, selective estrogen receptor modulators (SERMs), have demonstrated potential cardioprotective effects. However, the underlying molecular mechanisms by which these SERMs modulate Transforming Growth Factor-β (TGF-β) signaling in human vascular smooth muscle cells (VSMCs) remain largely unexplored. This study sought to investigate the impact of tamoxifen and raloxifene on TGF-β-induced CHSY1 expression and Smad2 linker region phosphorylation in VSMCs and to elucidate the role of reactive oxygen species (ROS), NADPH oxidase (NOX), and kinase pathways in mediating these effects.Employing a comprehensive experimental strategy, VSMCs were treated with TGF-β in the presence or absence of tamoxifen, raloxifene, and various pharmacological inhibitors. Subsequently, CHSY1 mRNA expression, Smad2C and Smad2L phosphorylation, ROS production, p47phox and ERK 1/2 phosphorylation were assessed. Our results revealed that tamoxifen and raloxifene significantly attenuated TGF-β-mediated CHSY1 mRNA expression and Smad2 linker region phosphorylation, without affecting the canonical TGF-β-Smad2C pathway. Furthermore, these compounds effectively inhibited ROS production, p47phox and ERK 1/2 phosphorylation, implicating the involvement of the TGF-β-NOX-ERK-Smad2L signaling cascade in their cardioprotective properties.This study provides a comprehensive understanding of the molecular mechanisms underlying the cardioprotective effects of tamoxifen and raloxifene in VSMCs, offering valuable insights for the development of targeted therapeutic strategies aimed at atherosclerosis prevention and the promotion of cardiovascular health.

Endothelin‐1 dependent expression of GAG genes involves NOX and p38 mediated Smad linker region phosphorylation

Endothelin‐1 (ET‐1) is implicated in the development of atherosclerosis and mediates glycosaminoglycan (GAG) chain hyperelongation on proteoglycans. Our aim was to identify the ET‐1‐mediated signalling pathway involving NADPH oxidase (NOX), p38 MAP kinsae and Smad2 linker region phosphorylation (phospho‐Smad2L) regulate GAG synthesising enzymes mRNA expression (C4ST‐1 and ChSy1) involved in GAG chains hyperelongation in human vascular smooth muscle cells (VSMCs). Signalling intermediates were detected and quantified by Western blotting and the mRNA levels of GAG synthesising enzymes were assessed by quantitative real‐time polymerase chain reaction (qRT‐PCR). ET‐1 treatment of human VSMCs resulted in an increase in phospho‐Smad2L level. The TGF‐β receptor antagonist, SB431542 and the mixed ETA and ETB receptor antagonist bosentan, inhibited ET‐1‐mediated phospho‐Smad2L level. In the presence of apocynin and diphenyleneiodonium chloride (DPI) (NOX inhibitors) and SB239063 (p38 inhibitor) ET‐1‐mediated phospho‐Smad2L levels were inhibited. The gene expression levels of GAG synthesising enzymes post‐ET‐1 treatment were increased compared to untreated controls (p < 0.01). The ET‐mediated the mRNA levels of these enzymes were blocked by the bosentan, SB431542, SB239063, DPI, apocynin and antioxidant N‐acetyl‐L‐cysteine (NAC). ET‐1‐mediated signalling to GAG synthesising enzymes gene expression occurs via transactivation‐dependent pathway involving NOX, p38 MAP kinsae and Smad2 linker region phosphorylation.

Thrombin Improves Diabetic Wound Healing by ERK-Dependent and Independent Smad2/3 Linker Region Phosphorylation.

Impaired wound healing is one of the most noteworthy features and troublesome complications of diabetes mellitus, which arouses a rising global health concern without potent remedies. Thrombin is the major hemostatic agent applied at wound healing initiation and recently gained therapeutic credits in later phases. However, a rare investigation achieved prolonged use of thrombin and probed the detailed mechanism. The objective of this study is to investigate the effects and mechanism of thrombin on diabetic skin wound healing. The effect of thrombin on fibroblast proliferation, α-SMA, and Collagen I expression was firstly studied in vitro by Cell Counting Kit 8 (CCK8) and western blotting. Then, the specific phosphorylation site of SMAD2/3 and their ERK1/2 dependence during thrombin treatment were assessed by western blotting for mechanism exploration. After that, full-thickness wound defects were established in diabetic male SD rats and treated with thrombin in the presence or absence of PD98059 to observe the in vivo effects of thrombin and to confirm its ERK dependence. We found that thrombin promoted fibroblast proliferation and their α-SMA and Collagen I production. Mechanistically, thrombin induced phosphorylation of Smad2 linker region (Ser245/250/255) through ERK1/2 phosphorylation but promoted phosphorylation of Smad3 linker region (Ser204) independent of ERK1/2. Histological results showed that thrombin facilitated wound healing by promoting α-SMA and Collagen I expression, which was not abolished by inhibiting ERK phosphorylation. Collectively, this study validated the therapeutic efficacy of thrombin on diabetic wound healing and identified both ERK-dependent and -independent Smad2/3 linker region phosphorylation as the essential signaling events in this process.

High glucose-induced Smad3 linker phosphorylation and CCN2 expression are inhibited by dapagliflozin in a diabetic tubule epithelial cell model.

Background: In the kidney glucose is freely filtered by the glomerulus and, mainly, reabsorbed by sodium glucose cotransporter 2 (SGLT2) expressed in the early proximal tubule. Human proximal tubule epithelial cells (PTECs) undergo pathological and fibrotic changes seen in diabetic kidney disease (DKD) in response to elevated glucose. We developed a specific in vitro model of DKD using primary human PTECs with exposure to high D-glucose and TGF-β1 and propose a role for SGLT2 inhibition in regulating fibrosis. Methods: Western blotting was performed to detect cellular and secreted proteins as well as phosphorylated intracellular signalling proteins. qPCR was used to detect CCN2 RNA. Gamma glutamyl transferase (GT) activity staining was performed to confirm PTEC phenotype. SGLT2 and ERK inhibition on high D-glucose, 25 mM, and TGF-β1, 0.75 ng/ml, treated cells was explored using dapagliflozin and U0126, respectively. Results: Only the combination of high D-glucose and TGF-β1 treatment significantly up-regulated CCN2 RNA and protein expression. This increase was significantly ameliorated by dapagliflozin. High D-glucose treatment raised phospho ERK which was also inhibited by dapagliflozin. TGF-β1 increased cellular phospho SSXS Smad3 serine 423 and 425, with and without high D-glucose. Glucose alone had no effect. Smad3 serine 204 phosphorylation was significantly raised by a combination of high D-glucose+TGF-β1; this rise was significantly reduced by both SGLT2 and MEK inhibition. Conclusions: We show that high D-glucose and TGF-β1 are both required for CCN2 expression. This treatment also caused Smad3 linker region phosphorylation. Both outcomes were inhibited by dapagliflozin. We have identified a novel SGLT2 -ERK mediated promotion of TGF-β1/Smad3 signalling inducing a pro-fibrotic growth factor secretion. Our data evince support for substantial renoprotective benefits of SGLT2 inhibition in the diabetic kidney.

MEKK3 Regulates Angiogenesis through Smad Signaling

Angiogenesis is one of the most essential events during embryonic development. The previous studies proved that MEKK3 (MAP3K3) is critical for angiogenesis. However, precisely how MEKK3 regulates the vascular development remains largely unknown. To investigate the impact of MEKK3 in vascular development in vivo and its role in normal physiological and pathological settings, we generated VE-Cadherin CreERT2;MEKK3 flox/+ (control) and VE-Cadherin CreERT2;MEKK3 flox/- (MEKK3 iEC -/-) mice. MEKK3 deletion was induced from postnatal day (P) 1 by administration of tamoxifen daily. The retinal angiogenesis model was used to analyze the blood vessel development in P7 and P12 mice. Whole-mount staining of P7 retinas in MEKK3 iEC -/- mutants showed a significant decrease in vascularized area and branch formation. In contrast, control pups, although only having one copy of the MEKK3 gene in endothelial cells, developed a complete vascular sheet at the same dose of tamoxifen, as did the untreated pups. At P12, MEKK3 iEC -/- mice showed defects in vessel remodeling and arterial patterning. These retinas had dilated and tortuous arteries losing the identical marker. This phenotype accorded with the pathology of AVM (arteriovenous malformation). We then cultured murine aortic rings in 3D matrigel and induced sprouting with VEGF. Administration of TGF-b receptor kinase inhibitor SB431542 to the MEKK3 iEC-/- aorta showed a partial rescue effect on the number of sprouts and vascularized area. It implicated that the vascular malformation in the MEKK3 iEC-/- mice was partially due to the elevated TGF-b signals in endothelial cells. We confirmed the result by staining the retinas with SMA and N-cadherin, which are the downstream genes in TGF-b pathway. It turned out that both SMA and N-cadherin were expressed stronger in MEKK3 iEC-/- retinas than in the control ones. Next, by in vitro studies, we found that endothelial cell activation by VEGF treatment led to rapid and strong phosphorylation of R-SMADs at the linker regions in the control endothelial cells. Such phosphorylation was severely impaired in the MEKK3 -/- endothelial cells. The same results were found in the activation of ERK1/2 and p38. Inhibition of ERK1/2 but not p38 MAPK in control endothelial cells greatly reduced the VEGF-induced phosphorylation of R-SMADs at their linker regions. Inhibition of both ERK1/2 and p38 did not further reduce the SMAD linker region phosphorylation compared to inhibition of ERK1/2. Thus, we determined ERK1/2 are responsible for the VEGF-induced, MEKK3-dependent phosphorylation of R-SMADs at the linker region. Finally, since Notch pathway is an evolutionarily conserved signaling , which directly affects angiogenesis, we used aortic ring model to test their interaction, and detected the gene expression in endothelial cells. Hey1 and Hey2 were over expressed in MEKK3 -/- endothelial cells. Knockdown of Hey1 and Hey2 could rescue the deficient angiogenesis. Knockdown of RBPJ did not affect MEKK3 mediated angiogenesis or induction of Hey1 and Hey2. The above data supported a model whereby MEKK3-mediated SMAD signaling induces HEY1 and HEY2 independently of canonical Notch activation. Disclosures No relevant conflicts of interest to declare.

Dusp1 modulates activin/smad2 mediated germ layer specification via FGF signal inhibition in Xenopus embryos

ABSTRACT Activin, a member of the transforming growth factor (TGF-β) superfamily, induces mesoderm, endoderm and neuro-ectoderm formation in Xenopus embryos. Despite several previous studies, the complicated gene regulatory network and genes involved in this induction await more elaboration. We identified expression of various fibroblast growth factor (FGF) genes in activin/smad2 treated animal cap explants (AC) of Xenopus embryos. Activin/smad2 increased fgf3/8 expression, which was reduced by co-injection of dominant negative activin receptor (DNAR) and dominant negative Fgf receptor (DNFR). Interestingly, activin/smad2 also increased expression of dual specificity phosphatase 1 (dusp1) which has been known to inhibit Fgf signaling. Dusp1 overexpression in dorsal marginal zone caused gastrulation defect and decreased Jnk/Erk phosphorylation as well as Smad1 linker region phosphorylation. Dusp1 decreased neural and organizer gene expression with increasing of endodermal and ventral gene expression in smad2 treated AC, indicating that dusp1 modulates germ layer specification. Dusp1 decreased neural gene expression in fgf8 treated AC, suggesting that Erk and/or Jnk phosphorylation may be involved in fgf8 induced neural induction. In addition, dusp1 decreased the reporter gene activities of activin response element (ARE) and increased it for bmp response element (BRE), indicating that dusp1 modulates two opposite morphogen signaling of dorsal (activin/Smad2) and ventral (bmp/Smad1) tracks, acting to fine tune the Fgf/Erk pathway.

Artemisinin inhibits glycosaminoglycan chain synthesizing gene expression but not proliferation of human vascular smooth muscle cells

Pleotropic growth factor, transforming growth factor (TGF)-β drives the modification and elongation of glycosaminoglycan (GAG) chains on proteoglycans. Hyperelongated GAG chains bind and trap lipoproteins in the intima leading to the formation of atherosclerotic plaques. We have identified that phosphorylation of Smad2 linker region drives GAG chain modification. The identification of an inhibitor of Smad2 linker region phosphorylation and GAG chain modification signifies a potential therapeutic for cardiovascular diseases. Artemisinin renowned for its potent anti-malarial effects possesses a broad range of biological effects. Our aim was to characterise the anti-atherogenic role of artemisinin in vascular smooth muscle cells (VSMCs). We demonstrate that TGF-β mediated Smad2 linker region phosphorylation and GAG chain elongation was attenuated by artemisinin; however, we observed no effect on VSMC proliferation. Our data demonstrates the potential for artemisinin to be developed as a therapy to inhibit the development of atherosclerosis by prevention of lipid deposition in the vessel wall without affecting the proliferation of VSMCs.

In Vitro Effects of Curcumin on Transforming Growth Factor-β-mediated Non-Smad Signaling Pathway, Oxidative Stress, and Pro-inflammatory Cytokines Production with Human Vascular Smooth Muscle Cells.

Transforming growth factor-β (TGF-β) induces pro-inflammatory cytokines expression including interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) and these cytokines are associated with the development of atherosclerosis. Curcumin has anti-atherogenic effects and anti-inflammatory properties in the vascular wall, but the relative mechanisms are almost unknown. In the present study, we investigate the effect of curcumin on modulating the pro-inflammatory action of TGF-β in human vascular smooth muscle cells (VSMCs) and its molecular mechanisms. Cultured VSMCs were seeded into several groups: a control group (untreated group), a group treated with TGF-β, and several groups treated with TGF-β plus inhibitors. The cells were pre-treated with diphenyleneiodonium chloride, DPI, (20 μM), curcumin (5, 10 and 20 μM) and N-Acetyl-L-Cysteine, NAC, (10 mM) and then TGF-β (5 ng/mL) was added to the culture medium. The mRNA levels of IL-6 and TNF-α were detected by quantitative Real-Time Polymerase Chain Reaction. For monitoring the Smad2 linker region phosphorylation (pSmad2L), the western-blotting technique was applied and reactive oxygen species (ROS) generation was measured by utilizing 2',7'-dichlorofluorescein diacetate-based assay. TGF-β increased the mRNA expression of IL-6 (p=0.02 and p=0.001) and TNF-α (p =0.014 and p = 0.001) in a time-dependent manner, ROS production (p=0.03) and Smad2L phosphorylation (p=0.015). Pre-treatment with curcumin, DPI and NAC inhibited TGF-β-induced IL-6 (p=0.04) and TNF-α (p=0.001) mRNA expression, Smad2L phosphorylation (p=0.02) and ROS production (0.03). Pharmacological inhibition by Curcumin blocks TGF-β-induced ROS production, Smad2L phosphorylation, and IL-6 and TNF-α mRNA expression in human VSMCs.

Smad linker region phosphorylation is a signalling pathway in its own right and not only a modulator of canonical TGF-β signalling.

Transforming growth factor (TGF)-β signalling pathways are intensively investigated because of their diverse association with physiological and pathophysiological states. Smad transcription factors are the key mediators of TGF-β signalling. Smads can be directly phosphorylated in the carboxy terminal by the TGF-β receptor or in the linker region via multiple intermediate serine/threonine kinases. Growth factors in addition to hormones and TGF-β can activate many of the same kinases which can phosphorylate the Smad linker region. Historically, Smad linker region phosphorylation was shown to prevent nuclear translocation of Smads and inhibit TGF-β signalling pathways; however, it was subsequently shown that Smad linker region phosphorylation can be a driver of gene expression. This review will cover the signalling pathways of Smad linker region phosphorylation that drive the expression of genes involved in pathology and pathophysiology. The role of Smad signalling in cell biology is expanding rapidly beyond its role in TGF-β signalling and many signalling paradigms need to be re-evaluated in terms of Smad involvement.

Mechanisms of PAR-1 mediated kinase receptor transactivation: Smad linker region phosphorylation.

Protease activated receptors (PARs) transactivate both epidermal growth factor receptors (EGFR) and transforming growth factor (TGF)-β receptors (TGFBR1) in vascular smooth muscle leading to the increased expression of genes (CHST11 and CHSY1) which are rate limiting for the enzymes that mediate hyperelongation of glycosaminoglycan (GAG) chains on the lipid-binding proteoglycan, biglycan. This is an excellent model to investigate mechanisms of transactivation as the processes are biochemically distinct. EGFR transactivation is dependent on the classical matrix metalloprotease (MMP) based triple membrane bypass mechanism and TGFBR1 transactivation is dependent on Rho/ROCK signalling and integrins. We have shown that all kinase receptor signalling is targeted towards phosphorylation of the linker region of the transcription factor, Smad2. We investigated the mechanisms of thrombin mediated kinase receptor transactivation signalling using anti-phospho antibodies and Western blotting and gene expression by RT-PCR. Thrombin stimulation of phospho-Smad2 (Ser 245/250/255) and of phospho-Smad2(Thr220) via EGFR transactivation commences quickly and extends out to at least 4h whereas transactivation via TGFBR1 is delayed for 120min but also persists for at least 4h. Signalling of thrombin stimulated Smad linker region phosphorylation is approximately equally inhibited by the MMP inhibitor, GM6001 and the ROCK inhibitor, Y27632, and similarly expression of CHST11 and CHSY1 is approximately equally inhibited by GM6001 and Y27632. The data establishes Smad linker region phosphorylation as a central target of all transactivation signalling of GAG gene expression and thus an upstream kinase may be a target to prevent all transactivation signalling and its pathophysiological consequences.

Effect of cyclin E overexpression on resistance to trastuzumab and phosphorylation of SMAD3 in HER2+ breast cancer.

e12006 Background: Trastuzumab, an effective treatment for HER2+ breast cancer, can be limited by development of resistance. Cyclin E (CCNE) overexpression was implicated in trastuzumab resistance, though the association is not well understood. We sought to explore a potential mechanism focusing on altered phosphorylation of the TGFβ signaling protein, SMAD3, in CCNE overexpressing HER2+ breast cancer cells, leading to trastuzumab resistance. Methods: A transcriptional activity cell array (TRACER) was used to examine the relationship between specific transcription factors and trastuzumab resistance in BT474 HER2+ human breast cancer cells. Immunoblotting examined phosphorylation patterns of SMAD3 in trastuzumab sensitive and resistant conditions. Effects of blocking CCNE overexpression with a CDK2 inhibitor were measured. To test the relative influence of SMAD3 and SMAD3 linker region phosphorylation on cell proliferation, BT474R2 cell lines were created overexpressing SMAD3 (BT474R2-SMAD3), a mutant construct blocking CCNE-mediated SMAD3 linker region phosphorylation (BT474R2-5M), and a control (BT474R2-Blank). In vitro and in vivo studies measured response to trastuzumab. Results: Resistance to trastuzumab was confirmed for BT474R2 cells, showing a markedly increased resistance to treatment when compared to the parental line. Network analysis of TRACER data identified decreased SMAD3 activity was central to resistance. Immunoblotting showed SMAD3 expression was significantly downregulated in BT474R2 cells (p &lt; 0.01). Noncanonical phosphorylation of SMAD3 was increased in CCNE-overexpressing cells. Blocking CCNE mediated noncanonical phosphorylation of SMAD3 with CDK2 inhibition resulted in decreased proliferation of BT474R2 cells. In vitro studies examining response to trastuzumab showed an increased sensitivity for BT474R2-5M cells. Findings were validated in NSG mice inoculated with BT474R2-5M cells that developed significantly lower tumor volumes (p &lt; 0.001) when treated with trastuzumab. Conclusions: These results identified a mechanism of CCNE-mediated trastuzumab resistance, regulated through noncanonical SMAD3 phosphorylation, which may be effectively treated with CDK2 inhibition to help enhance the efficacy of trastuzumab for patients with HER2+ breast cancer.

Transforming growth factor-β1 mediated CHST11 and CHSY1 mRNA expression is ROS dependent in vascular smooth muscle cells.

Transforming growth factor (TGF)-β1 mediates glycosaminoglycan (GAG) chain hyperelongation on secreted proteoglycans and these modifications are associated with increased lipid binding in the vessel wall and the development of atherosclerosis. In vascular smooth muscle cells (VSMCs), TGF-β1 regulated GAG elongation via extracellular signal-regulated kinase (ERK) and p38 as well as Smad2 linker region phosphorylation. In this study, our aim was to identify the TGF-β1 mediated signalling pathway involving reactive oxygen species (ROS) and Smad2 linker region phosphorylation that regulate the mRNA expression of GAG synthesizing enzymes, chondroitin 4-O-sulfotransferase 1 (CHST11) and chondroitin sulfate synthase 1 (CHSY1) which are the rate limiting enzymes involved in GAG chain elongation. Signalling molecules were assessed by western blotting, quantitative real-time PCR was used for analysis of gene expression and intracellular ROS level was measured by a fluorescence based assay. TGF-β1 induced ROS production in VSMCs. Nicotinamide adenine dinucleotide phosphate oxidase (Nox) inhibitors, diphenyleneiodonium (DPI) and apocynin blocked TGF-β1 mediated Smad2 linker region phosphorylation. TGF-β1 treatment increased the mRNA levels of CHST11 and CHSY1. Pharmacological inhibition of Nox blocked TGF-β1 mediated mitogen activated protein kinases (MAPKs) phosphorylation and TGF-β1 stimulated CHST11 and CHSY1 mRNA expression. These findings demonstrated that TGF-β1 mediated expression of CHST11 and CHSY1 can occur via Nox-dependent pathways and Smad2 linker region phosphorylation.

Individual Smad2 linker region phosphorylation sites determine the expression of proteoglycan and glycosaminoglycan synthesizing genes

Growth factors such as thrombin and transforming growth factor (TGF)-β facilitate glycosaminoglycan (GAG) chain hyperelongation on proteoglycans, a phenomenon that increases lipoprotein binding in the vessel wall and the development of atherosclerosis. TGF-β signals via canonical carboxy terminal phosphorylation of R-Smads and also non-canonical linker region phosphorylation of R-Smads. The G protein coupled receptor agonist, thrombin, can transactivate the TGF-β receptor leading to both canonical and non-canonical Smad signalling. Linker region phosphorylation drives the expression of genes for the synthesis of the proteoglycan, biglycan. Proteoglycan synthesis involves core protein synthesis, the initiation of GAG chains and the subsequent elongation of GAG chains. We have explored the relationship between the thrombin stimulated phosphorylation of individual serine and threonine sites in the linker region of Smad2 and the expression of GAG initiation xylosyltransferase-1 (XT-1) and GAG elongation chondroitin 4-sulfotransferase-1 (C4ST-1) and chondroitin synthase-1 (CHSY-1) genes. Thrombin stimulated the phosphorylation of all four target residues (Thr220, Ser245, Ser250 and Ser255 residues) with a similar temporal pattern – phosphorylation was maximal at 15 min (the earliest time point studied) and the level of the phospho-proteins declined thereafter over the following 4 h. Jnk, p38 and PI3K, selectively mediated the phosphorylation of the Thr220 residue whereas the serine residues were variously phosphorylated by multiple kinases. Thrombin stimulated the expression of all three genes – XT-1, C4ST-1 and CHSY-1. The three pathways mediating Thr220 phosphorylation were also involved in the expression of XT-1. The target pathways (excluding Jnk) were involved in the expression of the GAG elongation genes (C4ST-1 and CHSY-1). These findings support the contention that individual Smad linker region phosphorylation sites are linked to the expression of genes for the initiation and elongation of GAG chains on proteoglycans. The context of this work is that a specific inhibitor of GAG elongation represents a potential therapeutic agent for preventing GAG elongation and lipid binding and the results indicate that the specificity of the pathways is such that it might be therapeutically feasible to specifically target GAG elongation without interfering with other physiological processes with which proteoglycans are involved.

Reactive Oxygen Species and p38MAPK Have a Role in the Smad2 Linker Region Phosphorylation Induced by TGF-β.

Transforming growth factor-β (TGF-β) in addition to the C-terminal region can phosphorylate receptor-regulated Smads (R-Smads) in their linker region. The aim of the present study was to evaluate the role of signaling mediators such as NAD(P)H oxidases (reactive oxygen species [ROS] generators), ROS, and ROS-sensitive p38 mitogen-activated protein kinase (p38MAPK) in this signaling pathway in cultured human vascular smooth muscle cells (VSMCs). The present in vitro study was performed on human VSMCs. Proteins were detected by western blotting utilizing an anti-phospho-Smad2 (Ser245/250/255) rabbit polyclonal antibody and a horseradish peroxidase-labeled secondary antibody. Glyceraldehyde-3-phosphate dehydrogenase was used as a loading control. The phospho-Smad2 linker region (pSmad2L) was detected in all the experimental groups: a control group (untreated group), a group treated with TGF-β (2 ng/mL), and a group treated with TGF-β plus different inhibitors. The data were normalized and presented as mean±SEM. The statistical analyses were performed using SPSS, version 16.0, and the nonparametric Kruskal-Wallis test. A P value smaller than 0.05 was considered statistically significant. The VSMCs treated with TGF-β (2 ng/mL) showed a time-dependent increase in the pSmad2L level. The highest level was observed at 15 minutes (P=0.03). The inhibitors of NAD(P)H oxidases (diphenyleneiodonium and apocynin) (P=0.04), ROS scavenger (N-acetylcysteine) (P=0.04), and p38MAPK inhibitor (SB-202190) (P=0.04) were able to reduce the increased level of the pSmad2L by TGF-β. Our results suggested that NAD(P)H oxidases played an important role in the Smad2L phosphorylation in the human VSMCs. Furthermore, our results confirmed that ROS and p38MAPK were involved in this signaling pathway. Thus, TGF-β via a ROS-dependent mechanism can transmit its signals to the pSmad2L.

Thrombin promotes PAI-1 expression and migration in keratinocytes via ERK dependent Smad linker region phosphorylation

Keratinocyte proliferation and migration is essential during re-epithelialisation for the restoration of the epithelial barrier during skin wound healing. Numerous growth factors are involved in the stimulation of keratinocyte proliferation and migration. The signalling pathways that drive these processes during wound healing are not well defined. This study investigated thrombin-mediated signalling in keratinocytes. The thrombin receptor, protease-activated receptor 1 (PAR-1) is a seven transmembrane G-protein coupled receptor that is known to transactivate the epidermal growth factor receptor (EGFR). Immortalized human keratinocytes (HaCaT cells) were treated with thrombin and selective inhibitors to EGFR and MAP kinases. Whole cell lysates were separated on SDS-PAGE and analysed by Western blot using antibodies against transcription factor Smad2. Quantitative real-time polymerase chain reaction was used to measure the mRNA expression of PAI-1 while scratch wound assays were used to measure keratinocyte migration. Western blot data showed that thrombin mediates PAR-1 transactivation of EGFR and the downstream phosphorylation of the transcription factor Smad2 linker (Smad2L) region. ERK1/2 inhibition by UO126 caused a decrease in Smad2L phosphorylation while the p38 inhibitor SB202190 and JNK inhibitor SP600125 did not. Smad2L Ser250 was specifically phosphorylated by this thrombin mediated pathway while Ser245 and Ser255 were not. Thrombin increased PAI-1 mRNA expression and keratinocyte migration and this was reduced when either EGFR or ERK1/2 were blocked. Taken together these results show that thrombin mediated mRNA expression of PAI-1 in keratinocytes and migration occurs via EGFR transactivation and involves signalling intermediates ERK1/2 and Smad2 and may be a key pathway in skin wound healing.

Flavopiridol Inhibits TGF-β-Stimulated Biglycan Synthesis by Blocking Linker Region Phosphorylation and Nuclear Translocation of Smad2.

Transforming growth factor-β (TGF-β) is a pleiotropic growth factor implicated in the development of atherosclerosis for its role in mediating glycosaminoglycan (GAG) chain hyperelongation on the proteoglycan biglycan, a phenomenon that increases the binding of atherogenic lipoproteins in the vessel wall. Phosphorylation of the transcription factor Smad has emerged as a critical step in the signaling pathways that control the synthesis of biglycan, both the core protein and the GAG chains. We have used flavopiridol, a well-known cyclin-dependent kinase inhibitor, to study the role of linker region phosphorylation in the TGF-β-stimulated synthesis of biglycan. We used radiosulfate incorporation and SDS-PAGE to assess proteoglycan synthesis, real-time polymerase chain reaction to assess gene expression, and chromatin immunoprecipitation to assess the binding of Smads to the promoter region of GAG Synthesizing genes. Flavopiridol blocked TGF-β-stimulated synthesis of mRNA for the GAG synthesizing enzymes, and chondroitin 4-sulfotransferase (C4ST-1), chondroitin sulfate synthase-1 (ChSy-1) and TGF-β-mediated proteoglycans synthesis as well as GAG hyperelongation. Flavopiridol blocked TGF-β-stimulated Smad2 phosphorylation at both the serine triplet and the isolated threonine residue in the linker region. The binding of Smad to the promoter region of the C4ST-1 and ChSy-1 genes was stimulated by TGF-β, and this response was blocked by flavopiridol, demonstrating that linker region phosphorylated Smad can pass to the nucleus and positively regulate transcription. These results demonstrate the validity of the kinases, which phosphorylate the Smad linker region as potential therapeutic target(s) for the development of an agent to prevent atherosclerosis.

The role of specific Smad linker region phosphorylation in TGF-β mediated expression of glycosaminoglycan synthesizing enzymes in vascular smooth muscle

Hyperelongation of glycosaminoglycan chains on proteoglycans facilitates increased lipoprotein binding in the blood vessel wall and the development of atherosclerosis. Increased mRNA expression of glycosaminoglycan chain synthesizing enzymes in vivo is associated with the development of atherosclerosis. In human vascular smooth muscle, transforming growth factor-β (TGF-β) regulates glycosaminoglycan chain hyperelongation via ERK and p38 as well as Smad2 linker region (Smad2L) phosphorylation. In this study, we identified the involvement of TGF-β receptor, intracellular serine/threonine kinases and specific residues on transcription factor Smad2L that regulate glycosaminoglycan synthesizing enzymes. Of six glycosaminoglycan synthesizing enzymes, xylosyltransferase-1, chondroitin sulfate synthase-1, and chondroitin sulfotransferase-1 were regulated by TGF-β. In addition ERK, p38, PI3K and CDK were found to differentially regulate mRNA expression of each enzyme. Four individual residues in the TGF-β receptor mediator Smad2L can be phosphorylated by these kinases and in turn regulate the synthesis and activity of glycosaminoglycan synthesizing enzymes. Smad2L Thr220 was phosphorylated by CDKs and Smad2L Ser250 by ERK. p38 selectively signalled via Smad2L Ser245. Phosphorylation of Smad2L serine residues induced glycosaminoglycan synthesizing enzymes associated with glycosaminoglycan chain elongation. Phosphorylation of Smad2L Thr220 was associated with XT-1 enzyme regulation, a critical enzyme in chain initiation. These findings provide a deeper understanding of the complex signalling pathways that contribute to glycosaminoglycan chain modification that could be targeted using pharmacological agents to inhibit the development of atherosclerosis.

Pathobiological mechanisms of peritoneal adhesions: The mesenchymal transition of rat peritoneal mesothelial cells induced by TGF-β1 and IL-6 requires activation of Erk1/2 and Smad2 linker region phosphorylation

Peritoneal adhesions, primarily caused by surgical procedures, are the leading cause of pelvic pain, bowel obstruction, and infertility. TGF-β1 and IL-6 have been found to be elevated in the peritoneal fluid of patients during/after abdominal surgery. However, it remains to be determined whether these cytokines interact and facilitate adhesion formation by promoting mesothelial to mesenchymal transition (MMT). In the present study, isolated rat peritoneal mesothelial cells were treated with TGF-β1 and/or IL-6 which elicited MMT as determined by morphologic and biochemical techniques. During this transition, cellular morphology changed from that of cobblestone polygonal cells to elongated/spindle-shaped fibroblast-like cells. There was decreased expression of genes characteristic of mesothelial cells, such as E-cadherin, and increased expression of genes characteristic of the myofibroblast phenotype, including α-smooth muscle actin and the EDA form of fibronectin, both of which appear to mediate the transfer of force to the extracellular matrix. Partial characterization of relevant signaling pathways identified Erk1/2 activation, which was enhanced by combined TGF-β1/IL-6 administration, as a crucial necessary factor in the transition. Erk1/2 activation as well as the phosphorylation of the linker region of Smad2 and MMT could be blocked by the MEK inhibitor, U0126, suggesting that such activation may be a potential pharmaceutical target to prevent MMT. In addition, the phenotypic transition could be prevented by hydrocortisone.

Effect of Protein Phosphatase 2Ac and Norcantharidin on Smad3 Linker Region Phosphorylation in HK-2 Cells

Effect of Protein Phosphatase 2Ac and Norcantharidin on Smad3 Linker Region Phosphorylation in HK-2 Cells

Transforming growth factor β-mediated site-specific Smad linker region phosphorylation in vascular endothelial cells.

Transforming growth factor (TGF)-β regulates the function of vascular endothelial cells and may be involved in endothelial dysfunction. The canonical TGF-β pathway involves TGF-β receptor-mediated carboxy-terminal phosphorylation of Smad2; however, TGF-β signalling also activates numerous serine/threonine kinases that phosphorylate Smad2 in its linker region. The expression of phosphorylated Smad linker proteins were determined following TGF-β stimulation in the absence and presence of different serine/threonine kinase inhibitors in vascular endothelial cells. Proteins were quantified by Western blotting using specific antibodies to individual phosphorylated Smad2 linker region residues. TGF-β mediated the phosphorylation of all four Smad2 linker region residues of interest. Erk and Jnk specifically phosphorylate Ser245 while all mitogen-activated protein kinases phosphorylate Ser250 and Ser255. Thr220 and Ser245 are phosphorylated by phosphoinositide 3 kinase (PI3K), while Ser255 was phosphorylated by the PI3K/Akt pathway. CDK and GSK-3 were shown to phosphorylate Thr220 and Ser245. TGF-β also mediated plasminogen activator inhibitor-1 gene expression that was attenuated by p38 and CDK inhibitors. TGF-β-mediated phosphorylation of individual serine/threonine sites in the linker region of Smad2 occurs in a highly specific manner by kinases. These phosphorylations provide an opportunity to further understand a therapeutically targeted and very specific signalling pathway in vascular endothelial cells.