PI3K/Akt pathway mediates high glucose-induced lipogenesis and extracellular matrix accumulation in HKC cells through regulation of SREBP-1 and TGF-β1

Previous studies have shown that high glucose stimulates renal SREBP-1 gene expression and increases renal tubular cells lipid metabolism, however, the mechanisms remain elusive. In the present study we demonstrated that PI3K/Akt pathway was activated in human renal proximal tubular cell line (HKC) exposed to high glucose accompanied with up-regulation of SREBP-1, TGF-β1, lipid droplets deposits and extracellular matrix production. Inhibition of PI3K/Akt pathway by chemical LY294002 or specific short hairpin RNA (shRNA) vector prevented SREBP-1 and TGF-β1 up-regulation, as well as ameliorated HKC cells lipogenesis and extracellular matrix accumulation. These findings indicate that PI3K/Akt pathway potentially mediates high glucose-induced lipogenesis and extracellular matrix accumulation in HKC cells.     

Phospho-mTOR: A novel target in regulation of renal lipid metabolism abnormality of diabetes

The activation of Akt has been proved to involve in the lipogenesis of diabetic nephropathy. However, it's still not clear whether mTOR, another main gene in PI3K/Akt pathway, is also involved in the renal lipogenesis of diabetes. In the present study, it was revealed that the phosphorylation of mTOR was up-regulated in the renal tubular cells of diabetic rats, followed by the over-expression of SREBP-1, ADRP and lipogenesis. Again, high glucose increased the expression of phospho-mTOR accompanied with SREBP-1 and ADRP up-regulation and lipid accumulation in HKC cells. Rapamycin, known as mTOR inhibitor, was used to inhibit the activation of mTOR, which prevented effectively high glucose-induced SREBP-1 up-regulation and lipogenesis in HKC cells. Furthermore, high glucose-stimulated HKC cells transfected with wild-type mTOR vector showed the enhanced SREBP-1 and lipid droplets, however, TE mTOR vector (kinase dead)-transfected HKC cells presented resistance to high glucose and decreased SREBP-1 expression and lipogenesis. These above data suggested that phospho-mTOR mediated lipid accumulation in renal tubular cells of diabetes and might be the potential targets for treating lipogenesis of diabetic nephropathy.     

SREBP-1基因过表达促进人肾小管上皮细胞甘油三酯形成

目的 SREBP-1重组质粒转染人肾小管上皮细胞(HKC)检测SREBP-1基因表达和细胞内脂滴的关系。方法体外培养人肾小管上皮细胞并随机分为空白对照组、pcDNA3.1空质粒对照组和pcDNA3.1-SC1重组质粒转染组,采用阳离子脂质体法将SREBP-1特异性质粒pcDNA3.1-SC1及pcDNA3.1空质粒转染到细胞内并培养48小时,半定量RT-PCR和Westernblot分析目标基因表达丰度的变化,并采用油红O染色检测细胞内脂滴。结果 pcDNA3.1-SC1重组质粒转染的细胞内SREBP-1mRNA表达呈现明显升高,扩增条带积分光密度值分别是空白对照组和阴性对照组的6.158倍和4.194倍,SREBP-1蛋白也出现明显上调,条带积分光密度值为3.092±0.254。空白对照组和pcDNA3.1阴性对照组细胞内均未见有红染脂滴颗粒,而pcDNA3.1-SC1重组质粒转染组中出现了清晰的红染颗粒。结论 SREBP-1表达可增加人肾小管上皮细胞脂肪合成证实HKC细胞中SREBP-1表达和脂滴形成之间存在有直接关系。     

Transforming growth factor-β1 regulation of laminin γ1 and fibronectin expression and survival of mouse mesangial cells

The transforming growth factor-beta (TGF-β) 1 is a mediator of extracellular matrix (ECM) gene expression in mesangial cells and the development of diabetic glomerulopathy. Here, we investigate the effects of TGF-β1 on laminin γ1 and fibronectin polypeptide expression and cell survival in mouse mesangial cells (MES-13). TGF-β1 (10 ng/ml) stimulates laminin-γ1 and fibronectin expression ~two-fold in a time-dependent manner (0–48 h). TGF-β1 treatment also retards laminin-γ1 mobility on SDS-gels, and tunicamycin, an inhibitor of the N-linked glycosylation, blocks the mobility shift. TGF-β1 increases the binding of laminin γ1 to WGA-agarose and the binding is abolished by tunicamycin suggesting that laminin γ1 is modified by N-linked glycosylation. TGF-β1 also elevates fibronectin glycosylation but its mobility is not altered. The degradation of laminin γ1 and fibronectin proteins is reduced by their glycosylation. In addition, TGF-β1 enhances mesangial cell viability and metabolic activities initially (0–24 h); however, eventually leads to cell death (24–48 h). TGF-β1 elevates pro-apoptotic caspase-3 activity and decrease cell cycle progression factor cyclin D1 expression, which parallels cell death. These results indicate that TGF-β1 plays an important role in ECM expression, protein glycosylation and demise of mesangial cells in the diabetic glomerular mesangium. (Mol Cell Biochem 278: 165–175, 2005)     

高脂喂养大鼠肾小管上皮细胞SREBP-1、TGF-β1、α-SMA的表达和细胞外基质沉积

目的:探讨高脂喂养对大鼠肾脏小管上皮细胞SREBP-1、TGF-β1、α-SMA表达和细胞外基质(ECM)的影响。方法:高脂饲料喂养大鼠12周后,油红O检测肾脏脂质沉积,Masson染色检测肾小管间质细胞外基质沉积,免疫组化、Western blot和原位杂交检测SREBP-1、TGF-β1、α-SMA和FN的表达。结果:高脂喂养后大鼠体重明显增加,血糖、甘油三酯和胰岛素均升高,油红O检测显示大鼠肾小管上皮细胞内出现明显脂滴。SREBP-1蛋白和mRNA在肾小管上皮细胞内表达,高脂组高于正常对照组,分别是正常组的1.88倍和1.85倍;TGF-β1和α-SMA也定位于肾小管上皮细胞胞浆并出现上调。Masson染色显示高脂喂养大鼠肾间质ECM沉积增多,纤维粘连蛋白FN检测也显示模型组表达强于对照组。结论:高脂饮食喂养可能通过上调肾脏小管上皮细胞SREBP-1表达使细胞内脂滴沉积,并进一步诱导TGF-β1、α-SMA合成而导致细胞外基质堆积。     

Role of sterol regulatory element-binding protein 1 in regulation of renal lipid metabolism and glomerulosclerosis in diabetes mellitus

Diabetic renal disease is associated with lipid deposits in the kidney. The purpose of our study was to determine whether there is altered regulation of the sterol regulatory element-binding proteins (SREBPs) in the diabetic kidney and whether SREBPs mediate the abnormal renal lipid metabolism and diabetic renal disease. In streptozotocin-induced diabetes in the rat, there were marked increases in SREBP-1 and fatty acid synthase (FAS) expression, resulting in increased triglyceride (TG) accumulation. Treatment of diabetic rats with insulin prevented the increased renal expression of SREBP-1 and the accumulation of TG. The role of hyperglycemia in the up-regulation of SREBP-1 was confirmed in renal cells cultured in a high glucose media. High glucose induced increased expression of SREBP-1a and -1c mRNA, SREBP-1 protein, and FAS, resulting in increased TG content. To determine a direct role for SREBP in mediating the increase in renal lipids and glomerulosclerosis, we studied SREBP-1a transgenic mice with increased renal expression of SREBP-1. The increase in SREBP-1 was associated with increased expression of FAS and acetyl CoA carboxylase, resulting in increased TG content, increased expression of transforming growth factor beta1 and vascular endothelial growth factor, mesangial expansion, glomerulosclerosis, and proteinuria. Our study therefore indicates that renal SREBP-1 expression is increased in diabetes and that SREBP-1 plays an important role in the increased lipid synthesis, TG accumulation, mesangial expansion, glomerulosclerosis, and proteinuria by increasing the expression of transforming growth factor beta and vascular endothelial growth factor.     

CCN3/CCN2 regulation and the fibrosis of diabetic renal disease

Prior work in the CCN field, including our own, suggested to us that there might be co-regulatory activity and function as part of the actions of this family of cysteine rich cytokines. CCN2 is now regarded as a major pro-fibrotic molecule acting both down-stream and independent of TGF-β1, and appears causal in the disease afflicting multiple organs. Since diabetic renal fibrosis is a common complication of diabetes, and a major cause of end stage renal disease (ESRD), we examined the possibility that CCN3 (NOV), might act as an endogenous negative regulator of CCN2 with the capacity to limit the overproduction of extracellular matrix (ECM), and thus prevent, or ameliorate fibrosis. We demonstrate, using an in vitro model of diabetic renal fibrosis, that both exogenous treatment with CCN3 and transfection with the over-expression of the CCN3 gene in mesangial cells markedly down-regulates CCN2 activity and blocks ECM over-accumulation stimulated by TGF-β1. Conversely, TGF-β1 treatment reduces endogenous CCN3 expression and increases CCN2 activity and matrix accumulation, indicating an important, novel yin/yang effect. Using the db/db mouse model of diabetic nephropathy, we confirm the expression of CCN3 in the kidney, with temporal localization that supports these in vitro findings. In summary, the results corroborate our hypothesis that one function of CCN3 is to regulate CCN2 activity and at the concentrations and conditions used down-regulates the effects of TGF-β1, acting to limit ECM turnover and fibrosis in vivo. The findings suggest opportunities for novel endogenous-based therapy either by the administration, or the upregulation of CCN3.     

Activation of extracellular signal-regulated kinase by TGF-β1 via TβRII and Smad7 dependent mechanisms in human bronchial epithelial BEP2D cells

Transforming growth factor-β1 (TGF-β1) can activate mitogen-activated protein kinases (MAPKs) in many types of cells. The mechanism of this activation is not well elucidated. Here, we explore the role of TGF-β/Smads signaling compounds in TGF-β1-mediated activation of extracellular signal-regulated kinase (ERK) MAPK in human papillomavirus (HPV)-18 immortalized human bronchial epithelial cell line BEP2D and the role of TGF-β1-induced phosphorylation of ERK in proliferation and apoptosis of BEP2D. The cell models of siRNA-mediated silencing of TGF-β receptor type II (TβRII), Smad2, Smad3, Smad4, and Smad7 were employed in this study. Our results demonstrate that TGF-β1 activates ERK in a time-dependent manner with a maximum effect at 60 min; overexpression of Smad7 increased this TGF-β1-mediated phosphorylation of the ERK; and siRNA-mediated silencing of TβRII, Smad3, Smad4, and Smad7 abrogated this effect. Moreover, we observed that overexpression of Smad7 restored TGF-β1-mediated ERK phosphorylation in Smad4 knockdown cells but not in TβRII knockdown cells. In BEP2D cells, TGF-β1 treatment effectively inhibited cells’ proliferation and induced their apoptosis. Pretreatment with U0126, an inhibitor of ERK1/2, significantly enhanced the TGF-β1-mediated antiproliferative and apoptosis induction effects in BEP2D cells. These data revealed that TβRII and Smad7 play the critical roles in TGF-β1-mediated activation of ERK; Smad3 and Smad4 can play an indirect role through up-regulating Smad7 expression; and TGF-β1-induced phosphorylation of ERK may participate in BEP2D cell proliferation and apoptosis regulation.     

The effect of TGF-β1 and Smad7 gene transfer on the phenotypic changes of rat alveolar epithelial cells

The aim of this study was to investigate whether transforming growth factor-β1 (TGF-β1) could induce alveolar epithelial-mesenchymal transition (EMT) in vitro, and whether Smad7 gene transfer could block this transition. We also aimed to elucidate the possible mechanisms of these processes. The Smad7 gene was transfected to the rat type II alveolar epithelial cell line (RLE-6TN). Expression of the EMT-associated markers was assayed by Western Blot and Real-time PCR. Morphological alterations were examined via phase-contrast microscope and fluorescence microscope, while ultrastructural changes were examined via electron microscope. TGF-β1 treatment induced a fibrotic phenotype of RLE-6TN with increased expression of fibronectin (FN), α-smooth muscle actin (α-SMA) and vimentin, and decreased expression of E-cadherin (E-cad) and cytokeratin19 (CK19). After transfecting the RLE-6TN with the Smad7 gene, the expression of the mesenchymal markers was downregulated while that of the epithelial markers was upregulated. TGF-β1 treatment for 48 h resulted in the separation of RLE-6TN from one another and a change into elongated, myofibroblast-like cells. After the RLE-6TN had been transfected with the Smad7 gene, TGF-β1 treatment had no effect on the morphology of the RLE-6TN. TGF-β1 treatment for 48 h resulted in an abundant expression of α-SMA in the RLE-6TN. If the RLE-6TN were transfected with the Smad7 gene, TGF-β1 treatment for 48 h could only induce a low level of α-SMA expression. Furthermore, TGF-β1 treatment for 12 h resulted in the degeneration and swelling of the osmiophilic multilamellar bodies, which were the markers of type II alveolar epithelial cells. TGF-β1 can induce alveolar epithelialmesenchymal transition in vitro, which is dependent on the Smads signaling pathway to a certain extent. Overexpression of the Smad7 gene can partially block this process     

Effects of TGF-betas and a specific antagonist on apoptosis of immature rat male germ cells in vitro

Massive apoptosis of pubertal male germ cells is important for the development of functional spermatogenesis in the adult testis. Although the trigger(s) for male germ cell loss at puberty remain undefined, we have hypothesized that transforming growth factor-betas (TGF-βs) play an active role. Here we demonstrate that the three mammalian TGF-β isoforms, TGF-β1, TGF-β2 and TGF-β3, induce distinct apoptosis of pubertal spermatogonia and spermatocytes in a dose-dependent manner. Induction of male germ cell death by activation of caspase-3 was most pronounced with TGF-β2 compared to TGF-β1 and TGF-β3. Furthermore, we found colocalization of activated caspase-3 with apoptotic protease-activating factor-1 (Apaf-1) in apoptotic germ cells, thus indicating the importance of the intrinsic mitochondrial pathway in TGF-β-induced apoptosis. The specificity of the TGF-β effects was proven by addition of recombinant latency-associated peptide against TGF-β1 (rLAP-TGF-β1) which completely abolished TGF-β1-induced and TGF-β3-induced germ cell apoptosis. Although TGF-β2-triggered germ cell death also was significantly reduced by rLAP-TGF-β1, inhibition was not maximal. Our results suggest that the three TGF-β isoforms induce apoptosis of pubertal male germ cells via the mitochondrial pathway in vitro and are thus likely candidates involved in the excessive first wave of apoptosis of male germ cells during puberty. Lutz Konrad and Marcel Munir Keilani contributed equally to this work.     

高糖对肾小管上皮细胞脂质代谢影响及罗格列酮的干预研究

目的探讨高糖环境中肾小管上皮细胞脂质代谢变化,以及罗格列酮对其干预作用。方法体外培养HKC细胞,随机分为正常糖组、高糖组、罗格列酮干预组,采用油红染色检测细胞内脂质,免疫细胞化学和WesternBlot检测固醇调节元件结合蛋白1(sterolregulatory element binding protein-1,SREBP-1)表达。结果与正常糖组比较,高糖培养的肾小管上皮细胞内出现明显脂滴;免疫细胞化学、WesternBlot检测发现固醇调节元件结合蛋白1表达于胞浆,12、24、48、72h固醇调节元件结合蛋白1前体和成熟体表达高糖组均高于正常对照组,差异有统计学意义,其中以48h表达量最大(前体2.334±0.045,成熟体1.082±0.040),罗格列酮组相对于高糖组,前体和成熟体表达均下降,脂滴形成有所减少。结论高糖可诱导肾小管上皮细胞固醇调节元件结合蛋白1前体和成熟体表达升高,进一步导致脂质合成增多;罗格列酮减少脂质合成可能是部分通过抑制固醇调节元件结合蛋白1表达而实现。     

Morphological Alteration of Golgi Apparatus and Subcellular Compartmentalization of TGF-β1 in Golgi Apparatus in Gerbils Following Transient Forebrain Ischemia

Golgi apparatus (GA) is a very important organelle involved in the metabolism of numerous proteins. TGF-β1 plays an important role in supporting neuronal survival after ischemic insults. Little is known, however, about the morphological alteration of GA and subcellular compartmentalization of TGF-β1 in brain after ischemia. Therefore, our present study was designed to check for GA morphological alterations and TGF-β1 subcellular localization. GA immunoreactivities were examined in the somatosensory cortex of gerbils after 10 min transient forebrain ischemia. Confocal Immunofluorographs of TGF-β1 and TGN38 were also taken. Results indicated that no fragmentation of GA was found in gerbils of norm, shams and 6, 24 and 72 h postocclusion, but some of the cortical cells showed fragmentation of GA in gerbils 7 days postocclusion. TGF-β1 was colocalized with TGN38, a marker molecule for the GA. We conclude that there was morphological alterations of GA and TGF-β1 was present in GA in the somatosensory cortex after 10 min ischemia.     

The effects of ACE inhibitor and angiotensin receptor blocker on clusterin and apoptosis in the kidney tissue of streptozotocin-diabetic rats

Our first aim was to determine the effects of secreted clusterin (sCLU) and nuclear clusterin (nCLU) in diabetic nephropathy. We also aimed to investigate the post-effects of angiotensin II blockage treatment on clusterin expression and to compare these with apoptosis. Five groups of Wistar albino rats were used: First group consisted of healthy controls; the second group included the untreated STZ-diabetics; 30 days of irbesartan or perindopril treated STZ-diabetics formed the third and the fourth groups, respectively; while the subjects receiving a combined treatment with irbesartan and perindopril for 30 days consisted the fifth group. TUNEL method for apoptosis and immunohistochemical staining for TGF-β1, α-SMA, clusterin-β and clusterin-α/β antibodies were performed. Apoptotic cells especially increased in the kidney tubuli of untreated diabetic group and on the contrary, a significant decrease was observed in the group that received a combined drug treatment. While sCLU was increased in the glomeruli and tubuli of the untreated diabetic group, it was decreased in all the treated groups. An increase in the nCLU immunoreactivity was observed in the podocytes, mesangial cells, and the injured tubule cells of the untreated diabetic group. nCLU immunopositive cells were decreased in all treated diabetic groups. In addition to this, the distribution of nCLU was similar to the distribution of apoptotic cells in the diabetic groups. Our results indicate that sCLU expression in diabetic nephropathy was induced due to renal tissue damage, and the nCLU expression increase in renal tubuli was related to apoptosis. Although irbesartan and perindopril prevented further renal injury in diabetes, a combined application of low-dose ACEI and AT1R blockers revealed more efficient measures, by means of renal damage prevention.     

Regulation of melanin synthesis by the TGF-β family in B16 melanoma cells

Effects of representative members of the transforming growth factor-β (TGF-β) family, TGF-β1, activin A and BMP-2, on melanin content and expression of pigment-producing enzymes were examined in B16 melanoma cells. Treatment with TGF-β1 or activin A but not with BMP-2 significantly decreased melanin content and expression of Tyrosinase and Tyrp-1, suggesting an inhibitory effect of TGF-β1 and activin A on melanin synthesis. TGF-β1 completely inhibited melanin synthesis induced by α-melanin stimulating hormone (α-MSH), whereas activin A only slightly did. As compared with parental B16 cells, the inhibitory effects of TGF-β1 and activin A on melanin content were relative smaller in B16 F10 cells, a subline of B16 cells that contain more pigment. The present study indicates that in addition to TGF-β, activin negatively regulates melanogenesis in the absence of α-MSH, but that the activity in the presence of α-MSH was slightly different between TGF-β and activin.     

PTEN ameliorates high glucose-induced lipid deposits through regulating SREBP-1/FASN/ACC pathway in renal proximal tubular cells

Phosphatase and tensin homology deleted on chromosome ten (PTEN) is a negative regulator of PI3K/Akt pathway, and here we investigated the effect of PTEN on lipogenesis in diabetic rats and high glucose-stimulated human renal proximal tubular cell line (HKC). Decreased PTEN and increased phospho-Akt were found in kidney of diabetic rats, and in vitro research revealed that high glucose attenuated PTEN expression in a time-dependent manner, concomitant with activation of Akt. Again, expression of PTEN significantly inhibited high glucose-caused increased phospho-Akt and lipogenic genes including SREBP-1, fatty acid synthase (FASN) and acetyl-CoA carboxylase (ACC). Furthermore, we confirmed inhibition of TGF-β1 pathway with SB431542 blocked the effect of high glucose on PTEN down-regulation, an increase in phospho-Akt and lipogenesis. These above data suggest that decreased PTEN mediates high glucose-induced lipogenesis in renal proximal tubular cells and TGF-β1 might be involved in PTEN down-regulation.     

Adenoviral transduction of hTGF-β1 enhances the chondrogenesis of bone marrow derived stromal cells

TGF-β1 plays a necessary and important role in the induction of chondrogenic differentiation of bone marrow stromal cells (BMSCs). In this study, porcine BMSCs were infected with a replication-deficient adenovirus expression vector carrying the hTGF-β1 gene. The transduced BMSCs were cultured as pelleted micromasses in vitro for 21 days, seeded onto disk-shaped PGA scaffolds for 3 days and subsequently implanted into the subcutaneous tissue of mice. BMSCs transduced with AdhTGF-β1 expressed and secreted more hTGF-β1 protein in vitro than those of the control group. Histological and immunohistological examination of the pellets revealed robust chondrogenic differentiation. Tissues made from cells transduced with AdhTGF-β1 exhibited neocartilage formation after 3 weeks in vivo. The neocartilage occupied 42 ± 5% of the total tissue volume which was significantly greater than that of the control group. Furthermore, there was extensive staining for sulfated proteoglycans and type II collagen in the AdhTGF-β1 group compared to controls, and quantification of GAG content showed significantly greater amounts of GAG in experimental groups. The results demonstrate that transfer of hTGF-β1 into BMSCs via adenoviral transduction can induce chondrogenic differentiation in vitro and enhance chondrogenesis in vivo.     

BRD7 suppresses the growth of Nasopharyngeal Carcinoma cells (HNE1) through negatively regulating β-catenin and ERK pathways

BRD7 is a novel gene which involved NPC in our lab. Our previous studies showed that BRD7 was expressed at high level in normal nasopharyngeal epithelial tissues, but at low level in nasopharyngeal carcinoma biopsies and cell lines. In these papers, we found that ectopic expression of BRD7 can decrease cell proliferation and capability to form colonies in soft agar. FCM (Flow cytometry) assay indicated that the cell cycle progression from G1 to S phase was inhibited and the expression of cyclinD1 was significantly decreased after being transfected with BRD7 in HNE1 cells (NPC cells). To further investigate the molecular mechanism of BRD7 suppression of NPC cells growth, the cDNA microarray was performed to detect difference in gene expression profile induced by BRD7. The results indicated that 21 genes expression were changed after being transfected with BRD7 and the differentially expressed gene including α-catenin, cyclinD1, E2F3 was confirmed by western-blot. Next, we found that even though no obvious changes of the total expression of β-catenin were observed, the accumulation of β-catenin in nucleus was blocked. In addition, it was found that the expression of β-catenin was up-regulated in the complex composed of β-catenin and α-catenin in HNE1 cells induction of BRD7. So, we concluded that over-expression of BRD7 increased the expression of α-catenin which “hold” β-catenin in the complex and inhibited its accumulating in nucleus. At last, we demonstrated the c-jun, p-MEK, and p-ERK1/2 expression were down-regulated, and the Ap-1 promoter activity was inactive after being transfected with BRD7. We also found that over-expression of BRD7 can inactivate the c-jun and p-ERK1/2 after being treated with EGF in HNE1 cells. These results indicated that BRD7 played a negative role in ERK1/2 pathway. Taken together, our present results provide new insights for BRD7 function to inhibit NPC cells growth through negative regulating β-catenin and ERK1/2 pathways.     

Growth inhibition induced by transforming growth factor-β1 in human oral squamous cell carcinoma

Oral squamous cell carcinoma (OSCC) is a world-wide health problem and its incidence accounts for 1.9–3.5% of all malignant tumors. Transforming growth factor beta/Smads (TGF-β/Smads) signaling pathway plays an important role in oncogenesis, but its function and molecular mechanisms in OSCC remain unclear. Expression of transforming growth factor-β receptor type II (TβRII) and Smad4 was studied by immunohistochemistry in 108 OSCC patients and 10 normal controls. Function and molecular mechanisms of TGF-β/Smads signaling pathway was then investigated in two human tongue squamous carcinoma cell lines with high and low metastasis (Tb and Tca8113) by RT-PCR, Western Blot, immunofluorescence, cell growth curve and flow cytometry (FCM), respectively. TβRII and Smad4 were significantly down-regulated in tumor tissues (with or without lymph node metastasis) compared to normal oral epithelium tissues (P < 0.05). TGF-β1 induced arrest of the cell cycle rather than cell death in Tca8113 and Tb cells, and this influence was mediated by the increasing the expression and changing the location of its downstream components of TGF-β1/Smads signaling pathway. TGF-β1 rapidly increased the expression of p15 and p21 in both Tca8113 and Tb cells. TGF-β1 did not increase p27 expression in Tca8113 cells, but p27 expression was increased in Tb cells. These indicated that TGF-β1 induced G₁ arrest of cell cycle through a different regulating pathway in Tb cells compared with Tca8113 cells. Thus, we conclude that TGF-β/Smads signaling pathway play a important role on cell growth and metastasis potential in OSCC. Xiumei Wang, Wenjing Sun, and Jing Bai contributed equally to this paper.     

Transforming growth factor-beta 1 in adipose derived stem cells conditioned medium is a dominant paracrine mediator determines hyaluronic acid and collagen expression profile

Conditioned medium from adipose derived stem cells (ADSC-CM) stimulates both collagen synthesis and migration of fibroblasts, and accelerates wound healing in vivo. Recently, the production and secretion of growth factors has been identified as an essential function of adipose-derived stem cells (ADSCs). However, the main soluble factor of ADSC-CM which mediates paracrine effects and its underlying mechanism has not been elucidated yet. In this study, we considered transforming growth factor-beta1 (TGF-β1) as a strong candidate for paracrine effect of ADSC-CM and investigated collagen synthesis and hyaluronic acid synthase (HAS) expression. After ADSC-CM addition, collagen type I, type III, HAS and hyaluronic acid (HA) expressions on human dermal fibroblasts (HDFs) were evaluated. Furthermore, to clarify effects of TGF-β1 as a paracrine mediator, TGF-β1 antibody and external supplementary TGF-β1 were treated to HDFs. Collagens type I, type III, HAS-1 and HAS-2 mRNA expressions of HDFs were greatly increased by ADSC-CM treatment, however there was no change in TGF-β1 antibody treated HDFs compared with non-treated control. These results strongly demonstrate that TGF-β1 plays an important role as a paracrine mediator of ECM synthesis. The fact that TGF-β1 contained in ADSC-CM not only accelerates collagen deposition but also increase hyaluronic acid synthesis of HDFs through HAS-1 and HAS-2 expression was also elucidated in this study. Therefore, ADSC-CM shows promise for the treatment of cutaneous wounds and accelerates granulation formation during healing process.