Hyaluronan attenuates transforming growth factor-beta1-mediated signaling in renal proximal tubular epithelial cells

Increased expression of hyaluronan (HA) has been associated with both acute renal injury and progressive renal disease, although the functional significance of this remains unclear. There is overwhelming evidence that transforming growth factor (TGF)-beta1 is critical to the development of progressive renal disease. Recent studies suggest an interaction between HA and TGF-beta signaling in cancer cell biology. The aim of this study was to examine the potential role of HA as a modulator of TGF-beta1 function in renal proximal tubular epithelial cells (PTC). Under resting conditions, co-localization of the principal receptor for HA, CD44, and both the TGF-beta type I and type II receptors was demonstrated by immunoprecipitation and western analysis and further confirmed by immunocytochemistry and confocal microscopy. Stimulation of PTC with TGF-beta1 led to increased synthesis of both type III and type IV collagen assessed by Western analysis. Addition of HA did not alter collagen synthesis, but abrogated TGF-beta1-mediated increase in type III and type IV collagen. This effect was blocked by the addition of a blocking antibody to CD44 and also by inhibition of MAP kinase kinase (MEK) activity. Furthermore HA decreased TGF-beta1 activation of a luciferase-SMAD responsive construct, and decreased translocation of SMAD4 into the cell nucleus. We have previously demonstrated an anti-migratory effect of TGF-beta1 in a scratch wounding model. As with HA antagonism of TGF-beta1 extracellular matrix generation, HA reduced the anti-migratory effect of TGF-beta1 in a CD44-dependent manner. In contrast to the effect of TGF-beta1 on collagen synthesis, which is SMAD-dependent, the anti-migratory effect of TGF-beta1 in this model is known to be dependent of activation of RhoA. In the presence of HA, TGF-beta1-mediated activation of RhoA was also abrogated in a CD44-dependent manner. The results suggest that co-localization of CD44 and TGF-beta receptors facilitate modulation of both SMAD and non-SMAD-dependent TGF-beta1-mediated events by HA. Our results therefore suggest that alteration of HA synthesis may represent an endogenous mechanism to limit renal injury.     

Inhibition of p38 mitogen-activated protein kinase and transforming growth factor-beta1/Smad signaling pathways modulates the development of fibrosis in adriamycin-induced nephropathy

Inflammation and fibrogenesis are the two determinants of the progression of renal fibrosis, the common pathway leading to end-stage renal disease. The p38 mitogen-activated protein kinase (MAPK) and transforming growth factor (TGF)-beta1/Smad signaling pathways play critical roles in inflammation and fibrogenesis, respectively. The present study examined the beneficial renoprotective effect of combination therapy using the p38 MAPK pathway inhibitor (SB203580) and a TGF-beta receptor I (ALK5) inhibitor (ALK5I) in a mouse model of adriamycin (ADR) nephrosis. The p38 MAPK and TGF-beta1/Smad2 signaling pathways were activated in ADR-induced nephropathy in a sequential time course manner. Two weeks after ADR injection, the combined administration of SB203580 (1 mg/kg/24 hours) and ALK5I (1 mg/kg/24 hours) markedly reduced p38 MAPK and Smad2 activities. Moreover, the co-administration of SB203580 and ALK5I to ADR-injected mice resulted in a down-regulation of total and active TGF-beta1 production, reduced myofibroblast accumulation, and decreased expression of collagen type IV and fibronectin. In these mice, retardation in the development of glomerulosclerosis and interstitial fibrosis was observed. In conclusion, although p38 MAPK and TGF-beta1/Smad signaling pathways are distinct they coordinate the progression of renal fibrosis in ADR nephrosis. The co-administration of a p38 MAPK inhibitor and an ALK5 inhibitor may have potential applications in the treatment of renal fibrosis.     

Reduced Smad3 protein expression and altered transforming growth factor-beta1-mediated signaling in cystic fibrosis epithelial cells.

Cystic fibrosis (CF) is a disease characterized by an aggressive inflammatory response in the airways. Given the antiinflammatory properties of transforming growth factor (TGF)-beta1, it was our goal to examine components of TGF-beta1-mediated signaling in both a cultured cell model and a mouse model of CF. A CF-related reduction of protein levels of the TGF-beta1 signaling molecule Smad3 was found in both of these model systems, whereas Smad4 levels were unchanged. Functional effects of reduced Smad3 expression are manifest in our cultured cell model, as reduced basal and TGF-beta1-stimulated levels of luciferase expression using the TGF-beta1-responsive reporter construct 3TP-Lux in the CF-phenotype cells compared with control cells. However, TGF-beta1-stimulated responses using the A3-Luc reporter construct were normal in both cell lines. These results suggest that select TGF-beta1-mediated signaling pathways are impaired in CF epithelial cells. This selective loss of Smad3 protein expression in CF epithelium may also influence inflammatory responses. Our data demonstrate that both CF-phenotype cells lacking Smad3 expression, and A549 cells expressing a dominant-negative Smad3, are unable to support TGF-beta1-mediated inhibition of either the interleukin (IL)-8 or the NOS2 promoter. We conclude that a CF-related reduction in Smad3 protein expression selectively alters TGF- beta1-mediated signaling in CF epithelium, potentially contributing to aggressive inflammatory responses.     

VSL#3 probiotics regulate the intestinal epithelial barrier in vivo and in vitro via the p38 and ERK signaling pathways

Probiotics can play a role in enhancing intestinal barrier function. However, the underlying mechanisms are not fully understood. The aim of this study was to examine the effects of VSL#3 probiotics on colonic epithelium permeability, tight junction protein expression and MAPKs signaling pathways in vivo and in vitro. In vivo, acute colitis was induced by administration of 3.5% dextran sodium sulfate for 7 days. Rats in two groups were treated with either 15 mg VSL#3 or placebo via a gastric tube once daily after induction of colitis. Tight junction protein expression and the MAPKs signaling pathways were studied by immunohistochemistry and immunoblotting. In vitro, HT-29 cells were exposed to TNF-α for up to 48 h with or without pre-treatment with a p38 MAPK inhibitor, an ERK inhibitor or a JNK inhibitor. Then tight junction proteins and the phosphorylation of MAPKs were examined in the presence or absence of VSL#3. In vivo, VSL#3 probiotics significantly ameliorated the disease activity index from Day 4 onward. In acute colitis rats, decreased expression of the tight junction proteins were observed, whereas VSL#3 therapy prevented these changes and increased the expression of phosphorylated p38 (P-p38), and of phosphorylated ERK (P-ERK). In vitro, tight junction proteins, P-p38 and P-ERK in the VSL#3 group were significantly higher than in the control and TNF-α groups. The p38 MAPK inhibitor and the ERK inhibitor could effectively prevent this effect. VSL#3 probiotics protected the epithelial barrier and increased the tight junction protein expression in vivo and in vitro by activating the p38 and ERK signaling pathways.     

Activation of transforming growth factor-beta1/p38/Smad3 signaling in stromal cells requires reactive oxygen species-mediated MMP-2 activity during bone marrow damage

Dose-escalated chemotherapy has proven utility in a variety of treatment settings, including preparative regimens before bone marrow or hematopoietic stem cell transplantation. However, the potential damage imposed by aggressive regimens on the marrow microenvironment warrants further investigation. In the present study, we tested the hypothesis that dose-escalated chemotherapy, with etoposide as a model chemotherapeutic agent, activates the transforming growth factor beta-1 (TGF-beta1) signaling pathway in bone marrow stromal cells. After high-dose etoposide exposure in vitro, Smad3 protein was phosphorylated in a time-and dose-dependent manner in marrow-derived stromal cells, coincident with the release of active and latent TGF-beta1 from the extracellular matrix. Phosphorylation was modulated by p38 kinase, with translocation of Smad3 from the cytoplasm to the nucleus subsequent to its phosphorylation. Etoposide induced activation of TGF-beta1 followed the generation of reactive oxygen species and required matrix metalloproteinase-2 (MMP-2) protein availability. Chemotherapy effects were diminished in MMP-2(-/-) knockout stromal cells and TGF-beta1 knockdown small interfering RNA-transfected stromal cells, in which phosphorylation of Smad3 was negligible after etoposide exposure. Stable transfection of a human MMP-2 cDNA into bone marrow stromal cells resulted in elevated phosphorylation of Smad3 during chemotherapy. These data suggest TGF-beta1/p38/Smad3 signaling cascades are activated in bone marrow stromal cells after dose-escalated chemotherapy and may contribute to chemotherapy-induced alterations of the marrow microenvironment.     

Effects of transforming growth factor-beta, transforming growth factor-alpha, and other growth factors on renal proximal tubule cells

Transforming growth factor (TGF)-alpha, epidermal growth factor (EGF), and insulin-like growth factor-1 (IGF-1) addition to quiescent, confluent monolayers of rabbit renal proximal tubule cells in primary culture stimulated [3H]thymidine incorporation. TGF-alpha and EGF promoted a 14-fold rise in thymidine incorporation over control levels with half-maximal responses at 2 x 10(-9) M. IGF-1 only promoted a 4-fold rise in thymidine incorporation compared with control values with a half-maximal response of 10(-8) M. Platelet-derived growth factor alone did not stimulate [3H]thymidine incorporation and did not potentiate the effects of EGF or IGF-1 on DNA synthesis, suggesting that platelet-derived growth factor is neither a competence nor a progression growth factor for renal proximal tubule cells. TGF-beta inhibited both baseline and EGF-stimulated [3H]thymidine incorporation after 48 hours of exposure but enhanced EGF-stimulated DNA synthesis at 24 hours. Morphologic evaluation with phase contrast microscopy, scanning, and transmission electron microscopy demonstrated that TGF-beta promoted a dramatic phenotypic transformation of the epithelial monolayer with migration and adhesion of the cells to form solid clusters of adherent cells. Quantitative morphometry demonstrated that this transformation developed 24 hours after TGF-beta exposure, was nearing completion after 48 hours of TGF-beta treatment, and correlated to TGF-beta related inhibition of EGF-induced DNA synthesis (r = -0.82, p less than 0.01). These results demonstrate that EGF and TGF-alpha are the most potent growth promoters for renal proximal tubule cells. IGF-1 is only a modest growth promoter, whereas platelet-derived growth factor has no effect either as a competence or progressive growth factor. TGF-beta inhibited EGF-induced DNA synthesis but only after observable phenotypic transformation of the cells. The degree of TGF-beta promoted transformation on renal tubule cells was highly correlative to th e antiproliferative effect of TGF-beta, suggesting that similar molecular components which promote this phenotypic transformation may also be critical in the antiproliferative effect of TGF-beta.     

Increased glomerular and tubular expression of transforming growth factor-beta1, its type II receptor, and activation of the Smad signaling pathway in the db/db mouse

Activation of the renal transforming growth factor-beta (TGF-beta) system likely mediates the excess production of extracellular matrix in the diabetic kidney. To establish the role of the TGF-beta system in type 2 diabetic nephropathy, we examined the intrarenal localization and expression of the TGF-beta1 isoform, the TGF-beta type II receptor, and the Smad signaling pathway in the 16-week-old db/db mouse, a genetic model of type 2 diabetes that exhibits mesangial matrix expansion, glomerular basement membrane thickening, and renal insufficiency that closely resemble the human disease. Compared with its nondiabetic db/m littermate, the db/db mouse showed significantly increased TGF-beta1 mRNA expression by in situ hybridization in both glomerular and tubular compartments. Likewise, TGF-beta1 protein, by immunohistochemical staining, was increased in both renal compartments, but the fractional expression of TGF-beta1 protein was less than that of the mRNA in the glomerulus. In situ hybridization and immunohistochemical staining for the TGF-beta type II receptor revealed concordant and significant increases of both mRNA and protein in the glomerular and tubular compartments of diabetic animals. Finally, immunohistochemistry showed preferential accumulation of Smad3 in the nuclei of glomerular and tubular cells in diabetes. The complementary technique of Southwestern histochemistry using a labeled Smad-binding element demonstrated increased binding of nuclear proteins to Smad-binding element, indicating active signaling downstream of the TGF-beta stimulus. We therefore propose that the TGF-beta system is up-regulated at the ligand, receptor, and signaling levels throughout the renal cortex in this animal model of type 2 diabetes. Our findings suggest that the profibrotic effects of TGF-beta may underlie the progression to glomerulosclerosis and tubulointerstitial fibrosis that characterize diabetic nephropathy.     

Progesterone-dependent release of transforming growth factor-beta1 from epithelial cells enhances the endometrial decidualization by turning on the Smad signalling in stromal cells

Endometrial decidualization results from the differentiationof stromal cells in an ovarian steroid-sensitive manner. Humanendometrial tissues obtained from fertile women at various stagesof the menstrual cycle were subjected to immunohistochemistryto localize the components of the transforming growth factor-beta(TGF-ß) system. TGF-ß receptor-I and -IIexpression was higher in stromal cells than in epithelial cellsduring the secretory phase while no such variation was observedduring the proliferative phase. The expression of phosphorylatedSmad3 (pSmad2/3), an activated form of a component of the TGF-ßsignalling pathway, and translocation of pSmad2/3 from the cytoplasmto the nucleus were more pronounced in secretory endometrium.In coculture of human endometrial epithelial with stromal cells,each isolated from the proliferative endometrium, administrationof progesterone stimulated decidualization as well as TGF-ßsignalling activation in stromal cells. Progesterone also significantlyelevated the concentration of TGF-ß1 in the coculturemedium. Careful manipulation of the coculture, i.e. selectiveaddition and omission of the cellular components, showed thatthis progesterone-induced increase in secretion of TGF-ß1come mainly from epithelial cells. Moreover, administrationof TGF-ß1 (10 ng/ml) directly to cultured stromalcells enhanced the expression of prolactin as well as pSamd2/3even without progesterone. Taken together, our present datasupport the notion that progesterone induces stromal decidualizationindirectly, i.e. by enhancing the expression and secretion ofTGF-ß1 from epithelial cells. The secreted, epithelial-derivedTGF-ß1 then acts on adjacent stromal cells, at leastin part, to turn on Smad signalling that could lead to stromaldecidualization.     

Interaction between the transforming growth factor-beta type II receptor/Smad pathway and beta-catenin during transforming growth factor-beta1-mediated adherens junction disassembly

The aim of the current study was to examine the influence of transforming growth factor (TGF)-beta 1 on proximal tubular epithelial cell-cell interaction, with particular emphasis on the regulation of adherens junction complex formation. Stimulation of the proximal tubular cell line HK-2 cells by TGF-beta 1 led to loss of cell-cell contact and disassembly of both adherens and tight junctional complexes. Adherens junction disassembly was associated with reduction of both Triton-soluble and Triton-insoluble E-cadherin, and an increase in detergent-soluble beta-catenin. Under these conditions, immunoprecipitation and Western analysis demonstrated decreased association of beta-catenin, both with E-cadherin, alpha-catenin, and the cell cytoskeleton. Confocal microscopy after immunostaining, showed decreased intensity of peripheral E-cadherin staining, and redistribution of beta-catenin expression to a perinuclear location. Tight junction disassembly was manifest by a reduction in the expression of Triton-soluble occludin and ZO-1 by Western analysis and their disassociation manifested by immunostaining and confocal microscopy. Loss of cell-cell contact and disassembly of adherens junctions were seen after addition of TGF-beta 1 to the basolateral aspect of the cells. Immunoprecipitation experiments demonstrated co-localization of E-cadherin, beta-catenin, and TGF-beta 1 RII in unstimulated cells. After TGF-beta 1 stimulation, the TGF-beta 1 RII no longer associated with either E-cadherin or beta-catenin. Dissociation of the adherens junction protein from the TGF-beta 1 receptor was associated with increased beta-catenin tyrosine phosphorylation and decreased threonine phosphorylation. Furthermore after receptor ligand binding, beta-catenin became associated with the TGF-beta 1-signaling molecules Smad3 and Smad4.     

Reactive oxygen species-mediated signaling pathways in angiotensin II-induced MCP-1 expression of proximal tubular cells

Angiotensin II (AngII) has pleiotropic effects, the most well known of which is the generation of reactive oxygen species (ROS) and chemokines in inflammatory lesions. Monocyte chemoattractant protein-1 (MCP-1) is considered a major chemokine in the pathogenesis of kidney diseases. We examined signaling pathways of AngII-induced MCP-1 expression and the role of ROS in the murine proximal tubular cells (mProx) using various inhibitors. Furthermore, we compared the signaling pathways between mProx and mesangial cells (MC). AngII-induced MCP-1 protein expression in mProx at 6 h was largely blocked by ROS (N-acetylcysteine; 82 +/- 14%), Ras (N-acetyl-S-trans,trans-farnesyl-L-cysteine; 82 +/- 13%), and nuclear factor-kappaB (NF-kappaB) (parthenolide; 89 +/- 7.9%) inhibitors. Both AT1 receptor (AT1R) (Olmesartan; 41 +/- 12%) and the AT2R (PD123319; 24 +/- 11%) antagonists partially blocked the MCP-1 expression. Furthermore, mitogen-activated protein kinase (MAPK) pathways were also implicated in this protein expression, but it is less dependent on ROS/Ras pathways. In MC, protein kinase (calphostin C; 84 +/- 2.8%) and NF-kappaB (89 +/- 1.4%) inhibitors attenuated acute AngII-induced MCP-1 expression stronger than ROS/Ras inhibitors (1.0 +/- 0.9/29 +/- 9.5%). MAPK pathways, especially p38 MAPK, were involved in MC more than in mProx. AT1R (69 +/- 8.6%) and AT2R (57 +/- 21%) antagonists also were blocked. We suggested that, although NF-kappaB activation has a critical role, signaling pathways are different between mProx and MC. ROS-mediated signaling in mProx may have more contribution to AngII-induced inflammatory responses than to those in MC.     

ERK1/2 and p38 regulate trophoblasts differentiation in human term placenta

Mitogen-activated protein kinases (MAPKs) control many cellular events from complex programmes, such as embryogenesis, cell differentiation and proliferation, and cell death, to short-term changes required for homeostasis and acute hormonal responses. However, little is known about expression and activation of classical MAPKs, extracellular signal-regulated kinase1/2 (ERK1/2) and p38 in human placenta. Therefore, we examined the expression of ERK1/2 and p38 in trophoblasts from human term placenta, and their implication in differentiation. In vitro , freshly isolated cytotrophoblast cells, cultivated in 10% fetal bovine serum (FBS), spontaneously aggregate and fuse to form multinucleated cells that phenotypically resemble mature syncytiotrophoblasts, that concomitantly produce human chorionic gonadotropin (hCG) and human placental lactogen (hPL). This study shows that the level of ERK1/2 and p38 decreases with increasing days of culture, to reach an undetectable level after 5 days of culture. Moreover, pretreatment of cells with an ERK1/2-specific inhibitor (PD98059) and/or a p38-specific inhibitor (SB203580) suppressed trophoblast differentiation. Our results also demonstrate that the p38 pathway is highly solicited as compared to the ERK1/2 pathway in the differentiation process. Furthermore, ERK1/2 and p38 are rapidly activated upon addition of FBS, but the activation of p38 is delayed compared to that of ERK1/2. In summary, this study showed that ERK1/2 and p38 pathways are essential to mediate initiation of trophoblast differentiation.     

转化生长因子-β/Smad信号对人横纹肌肉瘤细胞RD生长和凋亡的调控

目的研究TGF-β／Smad信号对人胚胎性横纹肌肉瘤细胞系RD生长和凋亡的调控作用。方法采用小分子RNA干扰(RNAi)技术,以阳离子脂质体为载体,将生物合成的发夹状短链RNA(shRNA)转染RD细胞,封闭Smad4的表达以阻碍内源性TGF-β／Smad信号的转导;采用逆转录一聚合酶链反应(RT-PCR)和Western blot检验shRNA干扰Smad4表达的效果;采用免疫荧光化学,通过激光共聚焦显微镜检测Smad4封闭前后RD细胞中Smad2／3表达位置的变化;应用四甲基偶氮唑盐(MTY)和。H-thymidine摄入实验检测阻碍内源性TGF-β／Smad信号转导和以外源性TGF-β1刺激这2种条件下RD细胞的生长状况;采用流式细胞术和荧光化学染色检测上述2种条件下RD细胞的凋亡情况。结果RNAi技术可有效封闭RD细胞中Smad4 mRNA和蛋白的表达;Smad4的封闭导致Smad2／3由胞质入核明显减少;内源性TGF-β／Smad信号转导受阻不但抑制RD细胞生长,并且诱导凋亡;外源性、较高浓度的TGF-β1可抑制RD细胞生长,但不诱导凋亡。结论TGF-β／Smad信号在一定的浓度范围内精细调控RD细胞的生长和凋亡。     

Serum amyloid A induces CCL20 secretion in mononuclear cells through MAPK (p38 and ERK1/2) signaling pathways

Although the serum levels of SAA had been reported to be upregulated during inflammatory/infectious process, the role of this acute-phase protein has not been completely elucidated. In previous studies, we demonstrated that SAA stimulated the production of TNF-alpha, IL-1beta, IL-8, NO, and ROS by neutrophils and/or mononuclear cells. Herein we demonstrate that SAA induces the expression and release of CCL20 from cultured human blood mononuclear cells. We also focus on the signaling pathways triggered by SAA. In THP-1 cells SAA promotes phosphorylation of p38 and ERK1/2. Furthermore, the addition of SB203580 (p38 inhibitor) and PD98059 (ERK 1/2 inhibitor) inhibits the expression and release of CCL20 in mononuclear cells treated with SAA. Our results point to SAA as an important link of innate to adaptive immunity, once it might act on the recruitment of mononuclear cells.