Expression of insulin-like growth factor-I and transforming growth factor-beta in hypokalemic nephropathy in the rat

BACKGROUND: Potassium deficiency (KD) in the rat retards body growth but stimulates renal enlargement caused by cellular hypertrophy and hyperplasia, which is most marked in the outer medulla. If hypokalemia persists, interstitial infiltrates appear and eventually fibrosis. Since early in KD insulin-like growth factor-I (IGF-I) levels in the kidney are elevated, suggesting that it may be an early mediator of the exaggerated renal growth, and as transforming growth factor-beta (TGF-beta) promotes cellular hypertrophy and fibrosis, we examined the renal expression of these growth factors in prolonged KD. METHODS: Rats were given a K-deficient diet or were pair fed or ad libitum fed a K-replete diet for 21 days. Growth factor mRNA levels were measured in whole kidney and protein expression localized by immunohistochemistry. RESULTS: KD rats weighed less than pair-fed controls, while the kidneys were 49% larger. Their serum IGF-I and kidney IGF-I protein levels were depressed, as were their IGF-I mRNA levels in liver, kidney, and muscle. These changes can largely be attributed to decreased food intake. In contrast, kidney IGF binding protein-1 (IGFBP-1) mRNA and TGF-beta mRNA levels were increased significantly. Histology of outer medulla revealed marked hypertrophy and adenomatous hyperplasia of the collecting ducts and hypertrophy of the thick ascending limbs of Henle with cellular infiltrates in the interstitium. Both nephron segments immunostained strongly for IGF-I and IGFBP-1, but only the nonhyperplastic enlarged thick ascending Henle limb cells immunostained for TGF-beta, which was strongly positive. Prominent interstitial infiltrates with ED1 immunostained monocytes/macrophages were present. CONCLUSIONS: These findings are consistent with a sustained role for IGF-I in promoting the exaggerated renal growth of KD and appear to be mediated through local trapping of IGF-I by the overexpressed IGFBP-1, which together with IGF-I can promote renal growth. The selective localization of TGF-beta to hypertrophied nonhyperplastic nephron segments containing IGF-I raises the possibility that TGF-beta may be serving to convert the mitogenic action of IGF-I into a hypertrophic response in these segments. It is also conceivable that TGF-beta may be a cause of the tubulointerstitial infiltrate. Finally, the low circulating IGF-I levels likely contribute to the impaired body growth.     

Chondrocytic differentiation of mesenchymal stem cells sequentially exposed to transforming growth factor-beta1 in monolayer and insulin-like growth factor-I in a three-dimensional matrix.

This study evaluated chondrogenesis of mesenchymal progenitor stem cells (MSCs) cultured initially under pre-confluent monolayer conditions exposed to transforming growth factor-beta1 (TGF-beta1), and subsequently in three-dimensional cultures containing insulin-like growth factor I (IGF-I). Bone marrow aspirates and chondrocytes were obtained from horses and cultured in monolayer with 0 or 5 ng of TGF-beta 1 per ml of medium for 6 days. TGF-beta 1 treated and untreated cultures were distributed to three-dimensional fibrin disks containing 0 or 100 ng of IGF-I per ml of medium to establish four treatment groups. After 13 days, cultures were assessed by toluidine blue staining, collagen types I and II in situ hybridization and immunohistochemistry, proteoglycan production by [35S]-sulfate incorporation, and disk DNA content by fluorometry. Mesenchymal cells in monolayer cultures treated with TGF-beta1 actively proliferated for the first 4 days, developed cellular rounding, and formed cell clusters. Treated MSC cultures had a two-fold increase in medium proteoglycan content. Pretreatment of MSCs with TGF-beta1 followed by exposure of cells to IGF-I in three-dimensional culture significantly increased the formation of markers of chondrocytic function including disk proteoglycan content and procollagen type II mRNA production. However, proteoglycan and procollagen type II production by MSC's remained lower than parallel chondrocyte cultures. MSC pretreatment with TGF-beta1 without sequential IGF-I was less effective in initiating expression of markers of chondrogenesis. This study indicates that although MSC differentiation was less than complete when compared to mature chondrocytes, chondrogenesis was observed in IGF-I supplemented cultures, particularly when used in concert with TGF-beta1 pretreatment.     

The effects of transforming growth factor β1, insulin-like growth factor 1 and leptin on the proliferation of fetal chondrocytes

One method to exogenously enhance the repair of articular cartilage is the local application of growth factors. This method is based on the chondrogenic effects of some agents and their potential ability to enhance cell migration. Human fetal chondrocytes were isolated and cultured. Their proliferation under the influence of different agents was microscopically evaluated. Fetal calf serum at 5% and 10% concentrations induced microscopically visible cell proliferation. Transforming growth factor beta one (5 and 10 ng/ml), insulin-like growth factor 1 (5 and 10 nmol/l) and leptin (1 and 2 ng/ml) accelerated proliferation of the cells towards the increasing gradient of the agents. Fibroblast growth factor beta (5 and 10 ng/ml), bone morphogenic protein two (10 ng/ml) and laminin (1 mg/ml) did not affect cell proliferation. This study suggests that different agents can play a role in the proliferation of fetal chondrocytes.     

Chemotactic response of osteoblast-like cells to transforming growth factor beta

Transforming growth factor beta (TGF-beta) was tested for its ability to stimulate a chemotactic response in two clonal rat osteosarcoma (ROS) cell lines, 17/2 and 25/1. TGF-beta stimulated dose-dependent chemotaxis in both cell lines. In serum-containing media, maximal response was seen at a concentration of 500 fg (10⁻¹⁵g)/mL for the ROS 17/2 cells and 25 fg/mL for the ROS 25/1 cells. In serum-free media, the maximal chemotactic response to TGF-beta occurred at 5 fg/mL for both the ROS 17/2 and 25/1 cells. TGF-beta was not mitogenic at these dosages. The results indicate that TGF-beta could act as a chemoattractant for osteogenic cells in both demineralized bone matrix induced osteogenesis and in normal bone remodeling.     

The effects of platelet-derived growth factor antagonism in experimental glomerulonephritis are independent of the transforming growth factor-beta system

Platelet-derived growth factor B-chain (PDGF-B)- and transforming growth factor beta (TGF-beta)-mediated accumulation of extracellular matrix proteins contributes to many progressive renal diseases. In vivo, specific antagonism of either PDGF-B or TGF-beta in experimental mesangioproliferative glomerulonephritis resulted in an almost complete inhibition of matrix protein accumulation, which suggests an interaction between signaling pathways of these two growth factors. Because nothing is known on the nature of this possible interaction, PDGF-B was antagonized in the rat anti-Thy 1.1 model of glomerulonephritis by use of specific aptamers and its effects on the TGF-beta system were investigated. Antagonism of PDGF-B led to a significant reduction of glomerular matrix accumulation compared with scrambled aptamer-treated nephritic controls. PDGF-B antagonism had no effect on the overexpression of glomerular TGF-beta mRNA, TGF-beta protein, or the expression of TGF-beta receptor type I and II mRNA. By immunohistology, it was possible to detect overexpression of the cytoplasmic TGF-beta signaling molecules Smad2 (agonistic) and Smad7 (antagonistic) in glomeruli of nephritic control rats which peaked on day 7 after disease induction, i.e., the peak of mesangial cell proliferation in this model. However, immunohistology and Western blot analysis again revealed no difference in the glomerular expression of both Smad proteins between PDGF-B antagonized and nonantagonized nephritic animals. In addition, no difference in the glomerular expression of phosphorylated Smad2 (P-Smad2) was detected between the differently treated nephritic groups. These observations suggest that the effects of PDGF-B antagonism are independent of TGF-beta in mesangioproliferative glomerulonephritides.     

Zeolite A increases proliferation, differentiation, and transforming growth factor beta production in normal adult human osteoblast-like cells in vitro.

Silicon in trace amounts enhances bone formation, and the silicon-containing compound zeolite A (ZA) increases eggshell thickness in hens. In the studies reported here, treatment of nearly homogeneous strains of normal human osteoblast-like cells for 48 h with ZA at 0.1-100 micrograms/ml induced a dose-dependent increase (r = 0.35, P < 0.001) in DNA synthesis (n = 31) to 162 +/- 16% (mean +/- SEM) of control and in the proportion of cells in mitosis (n = 4) from 9.1 +/- 1.8 to 27.0 +/- 4.5% (r = 0.69, P < 0.005). ZA treatment also increased alkaline phosphatase activity (P < 0.05) and osteocalcin release (P < 0.05) but did not significantly affect collagen production per individual cell. The mitogenic action of ZA was dependent on cell seeding density over the range of 1250-40,000 cells per cm2, which is consistent with induction of an autocrine factor(s). TGF-beta is a potent mitogen for osteoblasts. ZA treatment increased the steady-state mRNA levels of transforming growth factor beta 1 (TGF-beta 1) and induced the release of the latent form of TGF-beta protein into the conditioned medium within 6 h. We conclude that ZA induces the proliferation and differentiation of cells of the osteoblast lineage.     

白细胞介素10对大鼠肝损伤时肝星状细胞转化生长因子β1 和血小板源生长因子基因表达的影响

目的探讨白细胞介素10(IL-10)在大鼠肝损伤过程中对肝星状细胞(HSC)表达转化生长因子β1(TGFβ1)和血小板源生长因子(PDGF)的影响.方法治疗组用含有IL-10基因的腺病毒载体通过尾静脉转染大鼠(n=11),同时设立空载体组(n=12)和空白对照组(n=12),另给以皮下注射四氯化碳,每周2次,8周后处死大鼠,离体肝脏用胶原酶灌注以及密度梯度离心方法分离HSC.应用酶联免疫吸附法测大鼠血清中IL-10水平,逆转录聚合酶链反应和蛋白印迹检测HSC的TGFβ1和PDGF表达.结果治疗组的IL-10水平明显高于空载体组和空白对照组(P<0.05);治疗组HSC的TGFβ1和PDGF的mRNA、蛋白表达水平均低于空载体组和空白对照组(P<0.01).结论 IL-10可以通过下调HSC的TGFβ1和PDGF的表达来减轻肝损伤时HSC的激活程度.     

Phosphatidylinositol 3-kinase translocates to the nucleus of osteoblast-like MC3T3-E1 cells in response to insulin-like growth factor I and platelet-derived growth factor but not to the proapoptotic cytokine tumor necrosis factor alpha.

Changes in the metabolism of nuclear inositides phosphorylated in the D3 position of the inositol ring, which may act as second messengers, mainly have been linked to cell differentiation. To clarify a possible role of this peculiar class of inositides also during cell proliferation and/or apoptosis, we have examined the issue of whether or not in the osteoblast-like clonal cell line MC3T3-E1 it may be observed an insulin-like growth factor-I (IGF-I)- and platelet-derived growth factor (PDGF)-dependent nuclear translocation of an active phosphatidylinositol 3-kinase (PI 3-K). We found that both the growth factors increased rapidly and transiently both the amount and the activity of immunoprecipitable nuclear PI 3-K. Intranuclear PI 3-K exhibited a massive tyrosine phosphorylation on the p85 regulatory subunit. Moreover, by means of coimmunoprecipitation experiments, we showed the presence, in isolated nuclei, of the p110beta catalytic subunit of PI 3-K. Enzyme translocation was blocked by the specific PI 3-K inhibitor LY294002. In contrast, intranuclear translocation of PI 3-K did not occur in response to the proapoptotic cytokine tumor necrosis factor alpha (TNF-alpha). IGF-I was able to counteract the apoptotic stimulus of TNF-alpha and this was accompanied by the intranuclear translocation of PI 3-K. LY294002 inhibited both intranuclear translocation of PI 3-K and the rescuing effect of IGF-I. These findings strongly suggest that an important step in the signaling pathways that mediate both cell proliferation and survival is represented by the intranuclear translocation of PI 3-K.     

Triiodothyronine stimulates45Ca-accumulation via synthesis of insulin-like growth factor-I in cultures of osteoblast-like cells

We examined the effects of triiodothyronine (T₃) on⁴⁵Ca-accumulation into extracellular matrix in osteoblast-like MC3T3-El cells cultured for 7-week period, which was considered to be long enough to develop calcification. T₃ increased⁴⁵Ca-accumulation into sodium dodecyl sulfate-insoluble, EDTA-extractable materials cultures of MC3T3-El cells in a dose-dependent manner in the range between 10 pM and 10 nM. Contrary to the stimulatory effect on⁴⁵Ca-accumulation, T₃ inhibited DNA synthesis in MC3T3-El cells dose dependently between 10 pM and 10 nM. T₃ stimulated the secretion of insulin-like growth factor-I (IGF-I) dose dependently between 0.1 nM and 10 nM in MC3T3-El cells cultured for 7 weeks. Antibodies to IGF-I suppressed the⁴⁵Ca-accumulation induced by T₃ almost to the control level. These results strongly suggest that T₃ stimulates⁴⁵Ca-accumulation via synthesis of IGF-I in osteoblast-like cells.     

Combined effects of insulin-like growth factor-1 and transforming growth factor-beta1 on periosteal mesenchymal cells during chondrogenesis in vitro

OBJECTIVE: Periosteum contains undifferentiated mesenchymal stem cells that have both chondrogenic and osteogenic potential, and has been used to repair articular cartilage defects. During this process, the role of growth factors that stimulate the periosteal mesenchymal cells toward chondrogenesis to regenerate articular cartilage and maintain its phenotype is not yet fully understood. In this study, we examined the effects of insulin-like growth factor-1 (IGF-1) and transforming growth factor-beta1 (TGF-beta1), alone and in combination, on periosteal chondrogenesis using an in vitro organ culture model. METHODS: Periosteal explants from the medial proximal tibia of 2-month-old rabbits were cultured in agarose under serum free conditions for up to 6 weeks. After culture the explants were weighed, assayed for cartilage production via Safranin O staining and histomorphometry, assessed for proliferation via proliferative cell nuclear antigen (PCNA) immunostaining, and assessed for type II collagen mRNA expression via in situ hybridization. RESULTS: IGF-1 significantly increased chondrogenesis in a dose-dependent manner when administered continuously throughout the culture period. Continuous IGF-1, in combination with TGF-beta1 for the first 2 days, further enhanced overall total cartilage growth. Immunohistochemistry for PCNA revealed that combining IGF-1 with TGF-beta1 gave the strongest proliferative stimulus early during chondrogenesis. In situ hybridization for type II collagen showed that continuous IGF-1 maintained type II collagen mRNA expression throughout the cambium layer from 2 to 6 weeks. CONCLUSION: The results of this study demonstrate that IGF-1 and TGF-beta1 can act in combination to regulate proliferation and differentiation of periosteal mesenchymal cells during chondrogenesis.     

Cyclic AMP stimulates45Ca-accumulation via synthesis of insulin-like growth factor-I in cultures of osteoblast-like cells

Summary The effects of cAMP on insulin-like growth factor-I (IGF-I) secretion and⁴⁵Ca-accumulation into extracellular matrix were investigated in cloned osteoblast-like MC3T3-E1 cells cultured for 7 weeks, which was considered to be long enough to develop calcification. Dibutyryl cAMP stimulated IGF-I secretion dose dependently in the range between 0.1 mM and 3 mM. Dibutyryl cAMP also stimulated accumulation of⁴⁵Ca into sodium dodecyl sulfate-insoluble, EDTA-extractable materials in cultures of these cells dose dependently between 0.1 mM and 3 mM. The patterns of both stimulations were similar. The dibutyryl cAMP-stimulated IGF-I secretion preceded the⁴⁵Ca-accumulation. Antibodies to IGF-I suppressed the dibutyryl cAMP-stimulated⁴⁵Ca-accumulation almost to the control level. Taking account of our previous report that exogenous IGF-I stimulates⁴⁵Ca-accumulation in cultures of these cells, the present results suggest that cAMP stimulates⁴⁵Ca-accumulation via synthesis of IGF-I in osteoblast-like cells.     

Blocking transforming growth factor-beta receptor signaling down-regulates transforming growth factor-beta1 autoproduction in keloid fibroblasts.

OBJECTIVE: To study transforming growth factor-beta1 (TGF-beta1) autoproduction in keloid fibroblasts and the regulation effect of blocking TGF-beta intracellular signaling on rhTGF-beta1 autoproduction. METHODS: Keloid fibroblasts cultured in vitro were treated with either rhTGF-beta1 (5 ng/ml) or recombinant adenovirus containing a truncated type II TGF-beta receptor gene (50 pfu/cell). Their effects of regulating gene expression of TGF-beta1 and its receptor I and II were observed with Northern blot. RESULTS: rhTGF-beta1 up-regulated the gene expression of TGF-beta1 and receptor I, but not receptor II. Over-expression of the truncated receptor II down-regulated the gene expression of TGF-beta1 and its receptor I, but not receptor II. CONCLUSIONS: TGF-beta1 autoproduction was observed in keloid fibroblasts. Over-expression of the truncated TGFbeta receptor II decreased TGF-beta1 autoproduction via blocking TGF-beta receptor signaling.     

Synergistic actions of platelet-derived growth factor and the insulin-like growth factors in vivo.

Topical application of growth factors has been shown to benefit both normal and impaired wound healing. In normal tissue repair, resident cells produce a "cocktail" of various types of growth factors that overlap in function. In vitro studies have proved that growth factor combinations can act synergistically to enhance cellular function beyond that achieved with individual growth factors. To determine whether similar combinations have a synergistic effect in vivo, we applied growth factor combinations topically to full-thickness skin wounds created in genetically diabetic mice. The C57BL/KsJ-db/db mouse is obese and has insulin-resistant diabetes, and it has been proved that this mouse has markedly impaired wound healing. Topical application of platelet-derived growth factor, insulin-like growth factor-I, or insulin-like growth factor-II enhances healing in this model. Marked synergism was found when platelet-derived growth factor and insulin-like growth factor-II were combined to produce augmentation in wound closure beyond that achieved by application of the individual growth factors. The synergistic effect allowed for improved tissue repair at doses of platelet-derived growth factor and insulin-like growth factor-II that were ineffective when applied individually. The addition of insulin-like growth factor-I or insulin to platelet-derived growth factor produced no significant synergism. Because multiple growth factors are released in the wound during the healing process, it is not surprising that their combination further enhances healing. Growth factor combinations should become an important addition to the armamentarium for the treatment of chronic, nonhealing wounds.     

Antisense extracellular signal-regulated kinase-2 gene therapy inhibits platelet-derived growth factor-induced proliferation, migration and transforming growth factor-beta(1) expression in vascular smooth muscle cells and attenuates transplant vasculopathy.

Platelet-derived growth factor-BB (PDGF-BB) enables vascular smooth muscle cells (VSMCs) to proliferate, migrate and secrete connective tissue matrix, which are critical events in transplant vasculopathy. However, little is known about the intracellular pathways that mediate these biologic responses of VSMCs. Extracellular signal-regulated kinase (ERK) pathway plays a major role in cellular responses and vascular diseases. In this study, we observed that the inhibition of ERK2 activity by recombinant adenovirus encoding antisense ERK2 (Adanti-ERK2) significantly suppressed the proliferation, converting of cell cycle from G(1) phase to S phase and directed migration, and partially abrogated transforming growth factor-beta(1) (TGF-beta(1)) expression in VSMCs stimulated with PDGF-BB. Ex vivo gene transfer of Adanti-ERK2 into rat aortic allograft attenuated chronic transplant vasculopathy by the inhibition of VSMC proliferation and migration. In conclusion, ERK2 is involved in PDGF-BB-induced VSMCs proliferation, migration and TGF-beta(1) expression and may be a potential therapeutic target for transplant vasculopathy.     

The effect of FK778 on acute rat renal allograft rejection and expression of platelet-derived growth factor and transforming growth factor-beta

BACKGROUND: Acute rejection is the single most important risk factor for the development of subsequent chronic allograft nephropathy (CAN). Both platelet-derived growth factor (PDGF) and transforming growth factor-beta (TGF-beta) are major mitogens mediating mesenchymal cell proliferation and epithelial to mesenchymal cell transition. Early posttransplant induction of these growth factors may start molecular mechanisms leading to CAN. A new promising immunosuppressive drug, FK778, is an analogue of the active metabolite of leflunamide, which inhibits de novo pyrimidine biosynthesis. Herein we investigated the effect of FK778 on acute rejection and on the expression of PDGF and TGF-beta both alone and in combination with cyclosporine (CsA) or tacrolimus (Tac). METHODS: Kidney transplantations were performed from Dark Agouti (DA) to Wistar-Furth (WF) rats with syngeneic controls between DA rats. No immunosuppression was given to syngeneic grafts. Allografts were immunosuppressed with FK778 alone or in combination with CsA or Tac. Grafts were harvested on day 5 for histology and immunohistochemistry (PDGF-A, -B, PDGFR-alpha, -beta, TGF-beta1, and TGF-betaR1). RESULTS: FK778 ameliorated the inflammatory response and reduced PDGF and TGF-beta expression in a dose-dependent manner. It also showed synergy with calcineurin inhibitors, an effect that was stronger with Tac than with CsA. CONCLUSIONS: Our results indicated that FK778 decreased PDGF and TGF-beta expression early in acute rejection, suggesting it to be a promising therapy for CAN.