Serine-204 in the linker region of Smad3 mediates the collagen-I response to TGF-ß in a cell phenotype-specific manner.

Regulation of TGF-ß1/Smad3 signaling in fibrogenesis is complex. Previous work by our lab suggests that ERK MAP kinase phosphorylates the linker region (LR) of Smad3 to enhance TGF-ß-induced collagen-I accumulation. However the roles of the individual Smad3LR phosphorylation sites (T179, S204, S208 and S213) in the collagen-I response to TGF-ß are not clear. To address this issue, we tested the ability of Smad3 constructs expressing wild-type Smad3 or Smad3 with mutated LR phosphorylation sites to reconstitute TGF-ß-stimulated COL1A2 promoter activity in Smad3-null or -knockdown cells. Blocking ERK in fibroblasts and renal mesangial cells inhibited both S204 phosphorylation and Smad3-mediated COL1A2 promoter activity. Mutations replacing serine at S204 or S208 in the linker region decreased Smad3-mediated COL1A2 promoter activity, whereas mutating T179 enhanced basal COL1A2 promoter activity and did not prevent TGF-ß stimulation. Interestingly, mutation of all four Smad3LR sites (T179, S204, S208 and S213) was not inhibitory, suggesting primacy of the two inhibitory sites. These results suggest that in these mesenchymal cells, phosphorylation of the T179 and possibly S213 sites may act as a brake on the signal, whereas S204 phosphorylation by ERK in some manner releases that brake. Renal epithelial cells (HKC) respond differently from MEF or mesangial cells; blocking ERK neither changed TGF-ß-stimulated S204 phosphorylation nor prevented Smad3-mediated COL1A2 promoter activity in HKC. Furthermore, re-expression of wild type-Smad3 or the S204A-Smad3 mutant in Smad3-knockdown HKC reconstituted Smad3-mediated COL1A2 promoter activity. Collectively, these data suggest that Serine-204 phosphorylation in the Smad3LR is a critical event by which ERK enhances Smad3-mediated COL1A2 promoter activity in mesenchymal cells.

Risk factors for cardiovascular disease in children on maintenance dialysis.

Cardiovascular disease mortality is high in children on maintenance dialysis, accounting for about 25% of patient deaths. Cardiovascular-related mortality rates for children on dialysis are higher than for children with successful kidney transplants. Data on the long-term consequences of risk factors for cardiovascular disease are lacking for pediatric end-stage renal disease patients. This article reviews pediatric data pertaining to the following risk factors: anemia, hypertension, hyperlipidemia, left ventricular hypertrophy, abnormal calcium-phosphorus metabolism, and hyperhomocysteinemia. The potential relationship of end-stage renal disease to the etiology of several functional disorders of the cardiovascular system is discussed. Clinical studies are needed to assess the prevalence of cardiovascular disease and of cardiovascular disease risk factors in the pediatric end-stage renal disease population. Possible preventive and therapeutic guidelines need to be developed for at-risk children on maintenance dialysis.

Sp1 and Smad proteins cooperate to mediate transforming growth factor-beta 1-induced alpha 2(I) collagen expression in human glomerular mesangial cells.

The mechanism(s) by which Smads mediate and modulate the transforming growth factor (TGF)-beta signal transduction pathway in fibrogenesis are not well characterized. We previously showed that Smad3 promotes alpha2(I) collagen gene (COL1A2) activation in human glomerular mesangial cells, potentially contributing to glomerulosclerosis. Here, we report that Sp1 binding is necessary for TGF-beta1-induced type I collagen mRNA expression. Deletion of three Sp1 sites (GC box) between -376 and -268 or mutation of a CAGA box at -268/-260 inhibited TGF-beta1-induced alpha2(I) collagen promoter activity. TGF-beta1 inducibility was also blocked by a Smad3 dominant negative mutant. Chemical inhibition of Sp1 binding with mithramycin A, or deletion of the GC boxes, inhibited COL1A2 activation by Smad3, suggesting cooperation between Smad3 and Sp1 in the TGF-beta1 response. Electrophoretic mobility shift assay showed that Sp1 and Smads form complexes with -283/-250 promoter sequences. Coimmunoprecipitation experiments demonstrate that endogenous Sp1, Smad3, and Smad4 form complexes in mesangial cells. In a Gal4-LUC reporter assay system, Sp1 stimulated the TGF-beta1-induced transcriptional activity of Gal4-Smad3, Gal4-Smad4 (266), or both. Using the transactivation domain B of Sp1 fused to the Gal4 DNA binding domain, we show that, in our system, the transcriptional activity of this Sp1 domain is not regulated by TGF-beta1, but it becomes responsive to this factor when Smad3 is coexpressed. Finally, combined Sp1 and Smad3 overexpression induces marked ligand-independent and ligand-dependent promoter activity of COL1A2. Thus, Sp1 and Smad proteins form complexes and their synergy plays an important role in mediating TGF-beta1-induced alpha2(I) collagen expression in human mesangial cells.

Shear stress enhances human endothelial cell wound closure in vitro.

Repair of the endothelium occurs in the presence of continued blood flow, yet the mechanisms by which shear forces affect endothelial wound closure remain elusive. Therefore, we tested the hypothesis that shear stress enhances endothelial cell wound closure. Human umbilical vein endothelial cells (HUVEC) or human coronary artery endothelial cells (HCAEC) were cultured on type I collagen-coated coverslips. Cell monolayers were sheared for 18 h in a parallel-plate flow chamber at 12 dyn/cm(2) to attain cellular alignment and then wounded by scraping with a metal spatula. Subsequently, the monolayers were exposed to a laminar shear stress of 3, 12, or 20 dyn/cm(2) under shear-wound-shear (S-W-sH) or shear-wound-static (S-W-sT) conditions for 6 h. Wound closure was measured as a percentage of original wound width. Cell area, centroid-to-centroid distance, and cell velocity were also measured. HUVEC wounds in the S-W-sH group exposed to 3, 12, or 20 dyn/cm(2) closed to 21, 39, or 50%, respectively, compared with only 59% in the S-W-sT cells. Similarly, HCAEC wounds closed to 29, 49, or 33% (S-W-sH) compared with 58% in the S-W-sT cells. Cell spreading and migration, but not proliferation, were the major mechanisms accounting for the increases in wound closure rate. These results suggest that physiological levels of shear stress enhance endothelial repair.

Sex steroids and the endothelium.

Gonadal steroids clearly influence the course of atherosclerotic cardiovascular disease in women. This observation has suggested that these hormones have beneficial effects on the physiology of the vascular wall. Increased arterial vascular caliber after estrogen treatment, decreased lipid levels in subjects receiving hormone replacement therapy, and the markedly decreased extent of atherosclerotic plaque formation in young women as compared with young men support a cardioprotective effect of ovarian steroids. Generally, it appears that the effects of 17beta-estradiol are particularly beneficial, and the mechanism of action is targeted largely to the endothelial cell. This review describes the evidence for positive effects of estrogens on endothelial cell biology and considers potential mechanisms for estrogen actions on endothelial cell signal transduction.

TGF-beta1 activates MAP kinase in human mesangial cells: a possible role in collagen expression.

BACKGROUND: Although the pathogenic relevance of transforming growth factor-beta (TGF-beta) to glomerular sclerosis has been established, the intracellular mechanisms by which TGF-beta induces extracellular matrix accumulation are not fully understood. We examined whether the mitogen-activated protein (MAP) kinase pathway is involved in TGF-beta1-induced collagen expression by cultured human mesangial cells. METHODS: The activation of MAP kinase pathways by TGF-beta1 was assessed by immunoblot with anti-phospho-ERK or -JNK antibodies and by transfection of plasmids expressing pathway-specific transcription activators fused to the DNA-binding domain of GAL4, as well as a GAL4 response element-luciferase reporter gene. The role of MAP kinase was assessed using biochemical inhibitors and transiently expressed dominant negative mutant constructs. The effects on TGF-beta1-induced alpha1(I) collagen expression were evaluated by Northern blot and by activation of a transiently transfected alpha1(I) promoter-luciferase reporter construct. RESULTS: ERK and JNK phosphorylation occurred 30 minutes and one hour, respectively, after TGF-beta1 treatment. A biochemical blockade of the ERK pathway inhibited TGF-beta1-induced alpha1(I) collagen expression. A dominant negative mutant of ERK1 but not of JNK decreased alpha1(I) gene promoter activation. Activation of the TGF-beta-responsive p3TP-Lux construct was partially inhibited by cotransfection of an ERK1 dominant negative mutant. CONCLUSION: These data indicate that MAP kinase pathways can be activated by TGF-beta1 in mesangial cells and that the ERK MAP kinase plays a role in TGF-beta-stimulated collagen I expression. Because we have shown previously that SMADs mediate TGF-beta1-stimulated collagen I expression, our findings raise the possibility of interactions between the MAP kinase and the SMAD pathways.

The transforming growth factor-beta/SMAD signaling pathway is present and functional in human mesangial cells.

BACKGROUND: Transforming growth factor-beta (TGF-beta) signals through a unique set of intracellular proteins, called SMADs, that have been characterized mainly in transient overexpression systems. Because several models of glomerulosclerosis suggest a role for TGF-beta in the extracellular matrix accumulation, we sought to characterize the role of SMAD proteins in mediating TGF-beta1 responses in a more physiological system using nontransformed human mesangial cells. METHODS: Endogenous SMAD expression and its modulation by TGF-beta1 were evaluated by Western and Northern blot analyses. Phosphorylation of Smad2 and Smad3 was determined by both phospholabeling and immunoblot. SMAD function and its role in type I collagen transcription were investigated in cotransfection experiments using promoter-luciferase reporter gene constructs. RESULTS: Cultured human mesangial cells express Smad2, Smad3, and Smad4 proteins. TGF-beta1 down-regulated Smad3 mRNA and protein expression, respectively, after 4 and 24 hours of treatment, whereas Smad2 and Smad4 were less affected. Both Smad2 and Smad3 were phosphorylated in response to TGF-beta1 beginning at 5 minutes, with maximal phosphorylation at 15 minutes, and decreasing phosphorylation by 2 hours. Smad2/3 and Smad4 coimmunoprecipitate only after TGF-beta1 treatment. The activity of a transiently transfected, TGF-beta-responsive construct, p3TP-Lux, was stimulated 3.6-fold by TGF-beta1. Overexpressed wild-type Smad3 increased basal luciferase activity, which was further stimulated by TGF-beta1. A dominant negative mutant form of Smad3 lacking the C-terminal serine phosphoacceptor sites (Smad3A) inhibited TGF-beta1-induced luciferase activity. TGF-beta1 also increased the activation of an alpha2(I) collagen promoter-luciferase reporter construct transfected into mesangial cells. This activation was inhibited by cotransfection with the Smad3A mutant. CONCLUSIONS: Smad2, Smad3, and Smad4 are present and activated by TGF-beta1 in human mesangial cells. The SMAD pathway is functional in these cells and appears to be involved in TGF-beta1-induced type I collagen gene transcription. These findings raise the possibility that SMAD signaling plays a role in glomerular matrix accumulation.

Angiostatin binds ATP synthase on the surface of human endothelial cells.

Angiostatin, a proteolytic fragment of plasminogen, is a potent antagonist of angiogenesis and an inhibitor of endothelial cell migration and proliferation. To determine whether the mechanism by which angiostatin inhibits endothelial cell migration and/or proliferation involves binding to cell surface plasminogen receptors, we isolated the binding proteins for plasminogen and angiostatin from human umbilical vein endothelial cells. Binding studies demonstrated that plasminogen and angiostatin bound in a concentration-dependent, saturable manner. Plasminogen binding was unaffected by a 100-fold molar excess of angiostatin, indicating the presence of a distinct angiostatin binding site. This finding was confirmed by ligand blot analysis of isolated human umbilical vein endothelial cell plasma membrane fractions, which demonstrated that plasminogen bound to a 44-kDa protein, whereas angiostatin bound to a 55-kDa species. Amino-terminal sequencing coupled with peptide mass fingerprinting and immunologic analyses identified the plasminogen binding protein as annexin II and the angiostatin binding protein as the alpha/beta-subunits of ATP synthase. The presence of this protein on the cell surface was confirmed by flow cytometry and immunofluorescence analysis. Angiostatin also bound to the recombinant alpha-subunit of human ATP synthase, and this binding was not inhibited by a 2,500-fold molar excess of plasminogen. Angiostatin's antiproliferative effect on endothelial cells was inhibited by as much as 90% in the presence of anti-alpha-subunit ATP synthase antibody. Binding of angiostatin to the alpha/beta-subunits of ATP synthase on the cell surface may mediate its antiangiogenic effects and the down-regulation of endothelial cell proliferation and migration.

Estradiol enhances endothelial cell interactions with extracellular matrix proteins via an increase in integrin expression and function.

Premenopausal women have a lower cardiovascular risk and a higher incidence of several autoimmune diseases involving blood vessels than men. Although the precise effects of estrogens on the cardiovascular system are largely unknown, recent data suggest that estrogens can exert direct regulatory effects on endothelial cells. In the present study, we show that 17beta-estradiol increases human umbilical vein endothelial cell attachment to the extracellular matrix proteins laminin-1, type IV collagen, type I collagen, and fibronectin. Estradiol enhanced adhesion most significantly to laminin-1 and to fibronectin-derived synthetic peptides containing an RGD sequence. Upon exposure to estradiol, an increase in beta1, alpha5 and alpha6 integrin mRNA was observed in subconfluent cells which was abrogated by treatment with cycloheximide. This increase was followed by a later enhancement in surface expression of the above integrins. In addition, integrin-mediated signaling was also enhanced by estrogens since an increase in tyrosine-phosphorylation of focal adhesion kinase induced by cell attachment was observed in estrogen-treated endothelial cells. Since integrins have an important role in mediating endothelial cell attachment, migration and differentiation, the increase in integrin expression and function induced by estradiol may be an important mechanism through which estrogens can promote neovascularization and vessel repair.

Basic fibroblast growth factor release by human coronary artery endothelial cells is enhanced by matrix proteins, 17beta-estradiol, and a PKC signaling pathway.

Endothelial cell function is regulated by interactions among cells, the extracellular matrix (ECM), and soluble mediators. We investigated this interaction by examining the effect of 17beta-estradiol (E2) on release of basic fibroblast growth factor (FGF-2) by human coronary artery endothelial cells (HCAEC) cultured on ECM proteins. After estrogen-depleted HCAEC were treated with E2 for 2 h, the conditioned media and cell layers were evaluated by immunoblot or ELISA for FGF-2. Release of FGF-2 into conditioned media was enhanced 10-fold compared to that on plastic and a further 2.4-fold by E2. As FGF-2 release from cells into the media increases, there is a corresponding decrease in the cellular content of FGF-2. By ELISA, FGF-2 release increased 406, 179, and 262%, on type IV collagen, laminin, or fibronectin, respectively. HCAEC cultured on type I collagen did not show E2-enhanced FGF-2 release by ELISA or immunoblot analysis. No changes were noted in HCAEC release of lactate dehydrogenase, tested as a control protein for cellular integrity. The estrogen receptor antagonist ICI182,780 blocked E2-induced, but not basal, FGF-2 release. Increased FGF-2 release occurred via a cycloheximide-insensitive pathway. Neither brefeldin-A nor genistein inhibited E2 enhancement of FGF-2 release by HCAEC cultured on fibronectin. However, the protein kinase C inhibitor calphostin C inhibited the E2-augmented FGF-2 release. These data show that E2 enhances FGF-2 release by HCAEC cultured on basement membrane proteins in the absence of wounding. This action requires the estrogen receptor and PKC activity, but does not require new protein synthesis, endoplasmic reticulum-to-Golgi-mediated secretion, or protein tyrosine phosphorylation. E2-enhanced FGF-2 release could contribute to the cardioprotective effects of estrogen.

Regulation of human mesangial cell collagen expression by transforming growth factor-beta1.

Transforming growth factor (TGF)-beta1 has been implicated in glomerular extracellular matrix accumulation. Since the spectrum and mechanism of changes in collagen turnover have not been fully characterized, we evaluated effects of TGF-beta1 on collagen expression by human mesangial cells. TGF-beta1 induced increased alpha1(I), alpha1(III), and alpha1(IV) collagen mRNA expression. Greater mRNA expression of matrix metalloproteinase (MMP)-2 was compensated by increased tissue inhibitor of metalloproteinases (TIMP)-2 mRNA. There was no change in TIMP-1 or membrane-type MMP mRNA expression, whereas MMP-1 mRNA decreased. Types I and IV collagen protein accumulated in both the cell layer and medium. Changes in collagen mRNA and protein occurred within 4 and 8 h, respectively. MMP-2 and TIMP-1 and -2 activities showed little change. Cycloheximide markedly decreased collagen detection within 4 h and reversed late, but not early, changes in alpha1(I) collagen mRNA. In this system, increased synthesis may be more significant than degradation for collagen accumulation, but collagen is short-lived in culture. Diverse TGF-beta1 actions on collagen turnover may be either immediate or mediated through synthesis of regulatory molecules.

Estrogen stimulates delayed mitogen-activated protein kinase activity in human endothelial cells via an autocrine loop that involves basic fibroblast growth factor.

BACKGROUND: Estrogen plays a significant role in protecting premenopausal women from cardiovascular disease. We have found that estradiol augments endothelial cell activities related to vascular healing and that human coronary artery and umbilical vein endothelial cells express estrogen receptors (ERs). Classically, the ER functions as a transcription factor, but the cytoplasmic targets of this genomic effect have not been defined for endothelial cells. In the present study, we examined the potential role of the mitogen-activated protein (MAP) kinases ERK1 and ERK2 as mediators of estrogen action. METHODS AND RESULTS: Human umbilical vein endothelial cells were estrogen depleted by culturing in hormone-free medium for 48 hours before experiments. 17Beta-estradiol (E2) stimulated a delayed (3 hours) 5- to 7-fold induction of ERK1/2 activity requiring activation of ER and new transcription/translation. Conditioned media from cells stimulated for 3 hours with E2 induced immediate ERK1/2 activation and phosphorylation of the basic fibroblast growth factor (bFGF) receptor. Moreover, ERK1/2 activation by E2 or by conditioned media was abrogated by treatment with neutralizing anti-bFGF antibody. CONCLUSIONS: These data describe an autocrine mechanism for E2 induction of ERK1/2 in HUVEC. Because our previous studies suggested that certain cardioprotective effects of estrogen are genomic in nature, the results are consistent with the hypothesis that autocrine stimulation of endothelial ERK1/2 activity by bFGF may play a role in the beneficial effects of estrogen on cardiovascular biology.

The nephrotic syndrome: from the simple to the complex.

The nephrotic syndrome represents a series of physiological results ensuing from the occurrence of significant urinary loss of protein. Although the common initiating event of proteinuria has long been established, the list of problems that result has continued to grow as our understanding of the pathophysiology of nephrosis has increased. This article discusses this pathophysiology, emphasizing the consequences of nephrotic-range proteinuria. These consequences include not only acute and subacute manifestations of disrupted homeostasis, but also ways in which nephrosis itself may amplify mechanisms by which progressive renal nephron loss occurs. New insights into the factors that initiate and maintain glomerulosclerosis and interstitial fibrosis may offer potential approaches to preventing or ameliorating chronic renal insufficiency.

Human prostate carcinoma cells express enzymatic activity that converts human plasminogen to the angiogenesis inhibitor, angiostatin.

Angiostatin is an inhibitor of angiogenesis and metastatic growth that is found in tumor-bearing animals and can be generated in vitro by the proteolytic cleavage of plasminogen. The mechanism by which angiostatin is produced in vivo has not been defined. We now demonstrate that human prostate carcinoma cell lines (PC-3, DU-145, and LN-CaP) express enzymatic activity that can generate bioactive angiostatin from purified human plasminogen or plasmin. Affinity purified PC-3-derived angiostatin inhibited human endothelial cell proliferation, basic fibroblast growth factor-induced migration, endothelial cell tube formation, and basic fibroblast growth factor-induced corneal angiogenesis. Studies with proteinase inhibitors demonstrated that a serine proteinase is necessary for angiostatin generation. These data indicate that bioactive angiostatin can be generated directly by human prostate cancer cells and that serine proteinase activity is necessary for angiostatin generation.

Increased expression of extracellular matrix proteins and decreased expression of matrix proteases after serial passage of glomerular mesangial cells.

The cellular events causing pathological extracellular matrix (ECM) accumulation in vivo are not well understood. Prolonged serial passage of several cell types in culture leads to increased production of extracellular matrix (ECM) proteins, but the mechanism for these putative fibrotic changes is not known. Here, human fetal glomerular mesangial cells were subjected to serial passage (P) in culture and the expression of ECM proteins, proteases and protease inhibitors was comprehensively evaluated. From P11 through P14, a series of phenotypic changes occurred. Steady-state expression of mRNA for alpha 1 chains of type III and type IV (but not type I) collagen, and for laminin beta 1 and gamma 1, increased 2- to 8-fold, while expression of mRNA for interstitial collagenase (MMP-1) and gelatinase A (MMP-2) virtually ceased. Expression of tissue-type plasminogen activator (tPA) mRNA also decreased markedly. Expression of mRNA for the tissue inhibitor of metalloproteinases (TIMP)-1, and of the smaller of two mRNA species for the PA inhibitor PAI-1, ceased by P14. There was a switch in expression of the two species of TIMP-2 mRNA: whereas the ratio of signal intensity comparing the 3.5 kb mRNA species to the 1.0 kb species was 5:1 up to P11, it was reversed (1:5) at P14 and later. Serial passage also led to changes in protein expression, with increased type IV collagen and laminin, but decreased interstitial collagenase and gelatinase A. The cells showed a progressive increase in staining for type IV collagen. These findings define the appearance of a matrix-accumulating phenotype in later-passage mesangial cells. Matrix expansion in vivo has been associated with increased transforming growth factor (TGF)-beta synthesis; the cells were found to show at least 5-fold increased expression of TGF-beta 1 mRNA from P8 to P16. However, treatment of P9 or P10 cells with graded doses of TGF-beta 1 increased expression of both collagen IV and gelatinase A mRNA and did not alter the ratio of signal intensity for TIMP-2 mRNA species. Thus, assumption of a matrix-accumulating phenotype by these cultured fetal glomerular mesangial cells is not accelerated by exogenous TGF-beta. These data describe an in vitro model of mesangial cell matrix turnover in which matrix accumulation could result from a concerted increase in ECM synthesis and decrease in ECM degradation.