Differential expression of thrombospondin and cellular fibronectin during remodeling in proliferative glomerulonephritis.

Thrombospondin-1 (TSP-1) and an alternatively spliced fibronectin (Fn)-EIIIA isoform are adhesive proteins associated with embryogenesis and tissue remodeling. We compared, by immunohistochemistry and in situ hybridization, the course of TSP-1 and Fn-EIIIA expression in a model of glomerulonephritis induced by Habu snake venom (HV) and characterized by mesangial cell migration, proliferation, and extracellular matrix (ECM) synthesis. At 24 hr after HV, TSP-1 and Fn-EIIIA proteins localized in the central aspects of lesions associated with platelets and macrophages and at the margins of lesions coinciding with mesangial cell migration (determined by Thy-1 staining). Mesangial cells at this time expressed TSP-1 but not Fn-EIIIA mRNA. TSP-1 protein and mRNA peaked in lesions at 48 hr and were associated with cell proliferation (determined by PCNA, alpha-smooth muscle actin phenotype, and expression of beta-PDGF receptor mRNA). TSP-1 expression declined at 72 hr when expression of ECM synthesis peaked, as determined by increased expression of collagen Type IV, laminin, and TGF-beta1 protein and mRNA. Mesangial cell expression of Fn-EIIIA was first observed at 48 hr and was most abundant at 72 hr after HV. Therefore, platelet- and macrophage-derived Fn-EIIIA and TSP-1 in early lesions are associated with mesangial cell migration. Mesangial cell upregulation of TSP-1 is associated with migration and proliferation but not maximal ECM accumulation, whereas mesangial cell expression of Fn-EIIIA is associated with proliferation and ECM accumulation. These results suggest distinctive temporal and spatial roles for TSP-1 and Fn-EIIIA in remodeling during glomerular disease. (J Histochem Cytochem 47:533-543, 1999)     

Control of cell proliferation via elevated NEDD8 conjugation in oral squamous cell carcinoma

Adenosine (ADO) is an intermediary metabolite of adenosine trisphosphate degradation and a vasoactive mediator. We showed previously that ADO induces contraction and proliferation in rat mesangial cells by a mechanism involving A1 and A2 receptors. The studies concerning the effect of ADO on extracellular matrix (ECM) accumulation in mesangial cells are scarce. The purpose of our study was to evaluate the effect of ADO and the effect of the selective stimulation of A1 and A2 ADO receptors on the expression of ECM components fibronectin and collagen type I, in human and rat renal mesangial cells. Cultured human and rat renal mesangial cells were subjected to selective stimulation of A1 and A2 ADO receptors for 24 and 48 h. Fibronectin and collagen type I expression was evaluated by Western blot; total collagen synthesis was measured by [³H]-proline incorporation into collagen proteins. ADO, A1 and A2 receptor stimulation induce increases in fibronectin expression in rat mesangial cells, and A1 receptor stimulation partially inhibits fibronectin expression in serum-stimulated rat mesangial cells, without any effect in human mesangial cells. A2 receptor stimulation reduces collagen type I expression in serum-stimulated mesangial cells. Neither ADO nor A1 or A2 receptor stimulation induce significant changes in total collagen synthesis. These data suggest that ADO is not a major regulator of ECM synthesis in rat and human mesangial cells.     

The inhibitory effects of endostatin on endothelial cells are modulated by extracellular matrix

We investigated the ability of extracellular matrix (ECM) proteins to modulate the response of endothelial cells to both promoters and inhibitors of angiogenesis. Using human dermal microvascular endothelial cells (HDMEC), we found that cells demonstrated different adhesive properties and proliferative responses to the growth factor VEGF depending upon which ECM protein with which they were in contact, with fibronectin having the most impact on VEGF-induced HDMEC proliferation and survival. More importantly, we observed that ECM could modulate the ability of the angiogenic inhibitor endostatin to prevent endothelial cell proliferation, survival and migration. We observed that growth on vitronectin or fibronectin impaired the ability of endostatin to inhibit VEGF-induced HDMEC proliferation to the greatest extent as determined by BrdU incorporation. We found that, following growth on collagen I or collagen IV, endostatin only inhibited VEGF-induced HDMEC proliferation at the highest dose tested (2500 ng/ml). In a similar manner, we observed that growth on ECM proteins modulated the ability of endostatin to induce endothelial cell apoptosis, with growth on collagen I, fibronectin and collagen IV impairing endostatin-induced apoptosis. Interestingly, endostatin inhibited VEGF-induced HDMEC migration following culture on collagen I, collagen IV and laminin, while migration was not inhibited by endostatin following HDMEC culture on other matrices including vitronectin, fibronectin and tenascin-C. These results suggest that different matrix proteins may affect different mechanisms of endostatin inhibition of angiogenesis. Taken together, our results suggest that the ECM may have a profound impact on the ability of angiostatic molecules such as endostatin to inhibit angiogenesis and thus may have impact on the clinical efficacy of such inhibitors.     

Collagenase expression and activity is modulated by the interaction of collagen types, hypoxia, and nutrition in human lung cells

Hypoxia not only controls organogenesis, embryogenesis, and wound repair, but also triggers tumor progression and metastasis. Matrix metalloproteinases (MMP), especially gelatinases (MMP-2, MMP-9) regulate the composition and stability of the extracellular matrix (ECM), which affects cell proliferation, migration, and differentiation. This study investigated the effect of hypoxia alone and in combination with ECM compounds and nutrition on MMP-2 and MMP-9 expression, activity, and synthesis in human lung fibroblasts and pulmonary vascular smooth muscle cells (VSMC). We also determined the expression of the tissue inhibitors of MMP (TIMP-1, -2). Cells were grown on plastic, collagen-I, collagen-IV, or gelatin and in either starving medium (0.1% serum) or growth medium (5% serum), and were subjected to normoxia or hypoxia (1% O(2)). Collagenases expression was determined by zymography. TIMP-1, -2 expression was assessed by Western blotting and RT-PCR. Depending on serum concentration human lung cells expressed pro-MMP-2 on all substrates. Hypoxia increased pro-MMP-2 expression, on collagen type I or type IV further via Erk1/2 and p38 MAP kinase signaling. MMP-9 was only expressed when cells were grown on collagen type IV and increased with serum concentration, and by hypoxia. TIMP-1 expression was only expressed when cells were grown on collagen type I and was significantly increased by hypoxia, while TIMP-2 expression was unchanged. We demonstrated that the hypoxia, ECM composition, and nutrition, rather than one of these conditions alone, modulate the expression and activity of collagenases and their inhibitors in primary human lung fibroblasts.     

Involvement of osteopontin upregulation on mesangial cells growth and collagen synthesis induced by intermittent high glucose

Glucose fluctuations are strong predictor of diabetic vascular complications. We explored the effects of constant and intermittent high glucose on the proliferation and collagen synthesis of cultured rat mesangial cells. Furthermore, the possible involvement of osteopontin (OPN) was assessed. In rat mesangial cells cultured in 5, 25, or 5 mmol/L alternating with 25 mmol/L glucose in the absence or presence of neutralizing antibodies to OPN, β3 integrin receptor and β5 integrin receptor, the cell proliferation, collagen synthesis, and the expression of OPN and type IV collagen were assessed. In cultured mesangial cells, treatment with constant or intermittent high glucose significantly increased [³H]thymidine incorporation in a time‐dependent manner. A modest increase was observed at 12 h, and further deteriorated afterwards, and reached the maximum incorporation at 48 h. Treatment with constant high glucose for 48 h resulted in significant increases in [³H]thymidine incorporation, cell number, [³H]proline incorporation, mRNA, and protein levels of type IV collagen and OPN compared with mesangial cells treated with the normal glucose, which were markedly enhanced in cells exposed to intermittent high glucose medium. In addition, neutralizing antibodies to either OPN or its receptor β3 integrin but not neutralizing antibodies to β5 integrin can effectively prevented proliferation and collagen synthesis of mesangial cells induced by constant or intermittent high glucose. Intermittent high glucose exacerbates mesangial cells growth and collagen synthesis by upregulation of OPN expression, indicating that glycemic variability have important pathological effects on the development of diabetic nephropathy, which is mediated by the stimulation of OPN expression and synthesis. J. Cell. Biochem. 109: 1210–1221, 2010. © 2010 Wiley‐Liss, Inc.     

Phorbol ester potentiates the growth inhibitory effects of troglitazone via up-regulation of PPARgamma in A549 cells

The activation of peroxisome proliferator-activated receptor gamma (PPARgamma) has been shown to induce growth arrest and differentiation of various cancer cells. In the current study, we investigated the effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on the expression of PPARgamma and proliferation of A549 cells. TPA elicited a dose- and time-dependent increase in PPARgamma mRNA and protein levels. PPARgamma expression in response to TPA was attenuated by pretreatment with bisindolylmaleimide I, N-acetyl-L-cysteine (NAC) and PD98059. TPA-induced protein kinase C (PKC) activation was linked to the generation of reactive oxygen species (ROS), both of which were indispensable for PPARgamma expression in A549 cells. Pretreatment with bisindolylmaleimide I or NAC blocked TPA-induced phosphorylation of extracellular signal-regulated kinase (ERK), suggesting that ERK-mediated signaling is also involved in the induction of PPARgamma. Furthermore, the growth inhibitory effect of troglitazone was significantly potentiated by prolonged incubation with TPA and was attenuated in the presence of GW9662, a specific inhibitor of PPARgamma. These effects were associated with an induction of cell cycle arrest at G0/G1 phase, which was accompanied by the induction of p21Waf1/Cip1 expression and decreased cyclin D1 expression. Taken together, these observations indicate that TPA synergizes with PPARgamma ligand to inhibit cell growth through up-regulation of PPARgamma expression.     

Overexpression of megsin induces mesangial cell proliferation and excretion of type IV collagen in vitro

Over-expression of megsin is associated with mesangial cell (MC) proliferation and extracellular matrix (ECM) accumulation. The underlying pathogenesis is unknown. This study demonstrate that over-expression of megsin induced incorporation of [³H]thymidine in MCs and PDGF-BB, TGF-β1 upregulation. Concentrations of PDGF-BB, TGF-β1 and type IV collagen in the culture medium of MCs transfected with megsin were higher than controls. Anti-PDGF-BB suppressed incorporation of [³H]thymidine in MCs transfected with megsin and mRNA expression of TGF-β1 in stable transformant MCs, suggesting that over-expression of megsin induces cell proliferation and ECM accumulation in MCs, upregulation of PDGF-BB and TGF-β1 is probably the main route involved in pathogenesis.     

Cooperativity between Sertoli cells and testicular peritubular cells in the production and deposition of extracellular matrix components

We examined the synthesis and deposition of extracellular matrix (ECM) components in cultures of Sertoli cells and testicular peritubular cells maintained alone or in contact with each other. Levels of soluble ECM components produced by populations of isolated Sertoli cells and testicular peritubular cells were determined quantitatively by competitive enzyme-linked immunoabsorbent assays, using antibodies shown to react specifically with Type I collagen, Type IV collagen, laminin, or fibronectin. Peritubular cells in monoculture released into the medium fibronectin (432 to 560 ng/microgram cell DNA per 48 h), Type I collagen (223 to 276 ng/microgram cell DNA per 48 h), and Type IV collagen (350 to 436 ng/microgram cell DNA per 48 h) during the initial six days of culture in serum-free medium. In contrast, Sertoli cells in monoculture released into the medium Type IV collagen (322 to 419 ng/microgram cell DNA per 48 h) but did not form detectable amounts of Type I collagen or fibronectin during the initial six days of culture. Neither cell type produced detectable quantities of soluble laminin. Immunocytochemical localization investigations demonstrated that peritubular cells in monoculture were positive for fibronectin, Type I collagen, and Type IV collagen but negative for laminin. In all monocultures most of the ECM components were intracellular, with scant deposition as extracellular fibrils. Sertoli cells were positive immunocytochemically for Type IV collagen and laminin but negative for fibronectin and Type I collagen. Co-cultures of peritubular cells and Sertoli cells resulted in interactions that quantitatively altered levels of soluble ECM components present in the medium. This was correlated with an increased deposition of ECM components in extracellular fibrils. The data correlated with an increased deposition of ECM components in extracellular fibrils. The data presented here we interpret to indicate that the two cell types in co-culture act cooperatively in the formation and deposition of ECM components. Results are discussed with respect to the nature of interactions between mesenchymal peritubular cell precursors and adjacent epithelial Sertoli cell precursors in the formation of the basal lamina of the seminiferous tubule.     

NHERF2 increases platelet-derived growth factor-induced proliferation through PI-3-kinase/Akt-, ERK-, and Src family kinase-dependent pathway

Platelet-derived growth factor (PDGF) has multiple functions including inhibition of apoptosis and promotion of cell proliferation. In this study, we show that Na(+)/H(+) exchanger regulatory factor 2 (NHERF2) binds to the carboxyl-terminal PDZ domain-binding motif of the PDGF receptor through a PDZ domain-mediated interaction, and evaluate the consequence on PDGF-induced proliferation. Stable transfection with NHERF2 increased the PDGF-induced phosphorylation of ERK and Akt in Rat1 embryonic fibroblasts. The phosphorylation of Akt was blocked by pretreatment with LY294002, a PI-3-kinase inhibitor, in both Rat1/NHERF2 and Rat1/vector cells. In Rat1/vector cells, PDGF-induced phosphorylation of ERK was completely inhibited by pretreatment with PD98059, a MEK inhibitor. In contrast, the NHERF2-dependent increase of ERK phosphorylation was not affected by pretreatment with PD98059 in Rat1/NHERF2 cells. Thus, the NHERF2-dependent increase of ERK phosphorylation occurs in a MEK-independent fashion. Pretreatment with PP2, a specific inhibitor of Src family tyrosine kinase, completely blocked the NHERF2-dependent increase of the phosphorylation of ERK and Akt, suggesting that NHERF2 up-regulates Erk phosphorylation through a Src family kinase-dependent pathway. Consistent with these results, the PDGF-induced thymidine incorporation was increased in Rat1/NHERF2 cells, and the NHERF2-dependent increase of thymidine incorporation was prevented by treatment with LY294002 and PP2 but not with PD98059. These results suggest that NHERF2 stimulates PDGF-induced proliferation by increasing PI-3-kinase/Akt, MEKindependent ERK, and Src family kinase-mediated signaling pathways.     

Activation of mitogen-activated protein kinase by oxidized low-density lipoprotein in canine cultured vascular smooth muscle cells

Oxidized low-density lipoprotein (OX-LDL) contributes significantly to the development of atherosclerosis. However, the mechanisms of OX-LDL-induced vascular smooth muscle cell (VSMC) proliferation are not completely understood. Therefore, we investigated the effect of OX-LDL on cell proliferation associated with a specific pattern of mitogen-activated protein kinase (MAPK) by [3H]thymidine incorporation and p42/p44 MAPK phosphorylation in canine cultured VSMCs. OX-LDL-induced [3H]thymidine incorporation and p42/p44 MAPK phosphorylation in a time- and concentration-dependent manner in VSMCs. Pretreatment of these cells with pertussis toxin (PTX) for 24 hours attenuated the OX-LDL-induced [3H]thymidine incorporation and p42/p44 MAPK phosphorylation, indicating that these responses were mediated through a receptor coupled to a PTX-sensitive G protein. In cells pretreated with PMA for 24 h and with either the PKC inhibitor staurosporine or the tyrosine kinase inhibitor genistein for 1h, substantially reduced the [3H]thymidine incorporation and p42/p44 MAPK phosphorylation in response to OX-LDL. Removal of Ca(2+) by addition of BAPTA/AM plus EGTA significantly inhibited OX-LDL-induced [3H]thymidine incorporation and p42/p44 MAPK phosphorylation, indicating the requirement of Ca(2+) for these responses. OX-LDL-induced [3H]thymidine incorporation and p42/p44 MAPK phosphorylation was completely inhibited by PD98059 (an inhibitor of MEK1/2) and SB203580 (an inhibitor of p38 MAPK). Furthermore, we also showed that overexpression of dominant negative mutants of Ras (RasN17) and Raf (Raf-301) completely suppressed MEK1/2 and p42/p44 MAPK activation induced by OX-LDL and PDGF-BB, indicating that Ras and Raf may be required for activation of these kinases. Taken together, these results suggest that the mitogenic effect of OX-LDL is mediated through a PTX-sensitive G-protein-coupled receptor that involves the activation o Ras/Raf/MEK/MAPK pathway similar to those of PDGF-BB in canine cultured VSMCs.     

Stretch-induced cell proliferation is mediated by FAK-MAPK pathway

Previously we reported that a uni-axial cyclic stretch treatment of rat 3Y1 fibroblasts induced focal adhesion kinase (FAK) tyrosine phosphorylation followed by mitogen-activated protein kinase (MAPK) activation (Wang et al., 2001) [Wang, J.G., Miyazu, M., Matsushita, E., Sokabe, M., Naruse, K., 2001. Uni-axial cyclic stretch induces focal adhesion kinase (FAK) tyrosine phosphorylation followed by mitogen-activated protein kinase (MAPK) activation. Biochem. Biophys. Res. Comm. 288, 356-361]. In the present study, we investigated whether stretch-induced MAPK activation leads to proliferation of fibroblasts. 3Y1 fibroblasts were subjected to a uni-axial cyclic stretch treatment (1 Hz, 120% in length) and the bromodeoxyuridine (BrdU) incorporation was measured to access cell proliferation. BrdU incorporation increased in a time-dependent manner and became significant within 6 hours. To investigate the involvement of FAK, we transiently expressed FAK mutants that lacked tyrosine phosphorylation site (s) (F397Y, F925Y, F397/925Y). Transient expression of wild-type FAK or mock vector did not inhibit the stretch-induced BrdU incorporation, however, the FAK mutants significantly blocked BrdU incorporation. Treatment of the cells with MAPK inhibitors, PD98059 or SB203580, blocked extracellular signal-regulated kinase (ERK) phosphorylation and p38 MAPK phosphorylation, respectively, and also blocked stretch-induced BrdU incorporation. These results suggest that the stretch-induced FAK activation followed by MAPK activation plays an important role in the stretch-induced proliferation of 3Y1 fibroblasts.     

Effects of epidermal growth factor on collagen expression by rat kidney mesangial cells in culture.

Increased collagen production by mesangial cells plays a key role in the development and progression of glomerular sclerosis. These changes reflect in part the impact of growth factors on mesangial cells. Since mesangial cells possess receptors for epidermal growth factor (EGF) and since previous studies have documented that EGF affects collagen synthesis in other cell types, we have examined the effects of EGF on collagen biosynthesis by rat kidney mesangial (RKM) cells in culture. Exposure for 24 h to EGF did not substantially affect the growth rate of RKM cells. While the types of collagen produced by RKM cells (types I, III, IV and V) were unaltered by exposure to EGF, total collagen production was reduced ( approximately 50%). This decrease in collagen expression was not uniform for each collagen type. Type I collagen production was inhibited by approximately 50%, both type III and type IV expression were each reduced by approximately 30%, but type V collagen production was suppressed by only approximately 15%. The reduction in type I collagen synthesis was accounted for mainly by a decrease in type I homotrimer production. Since type I molecules represent approximately 95% of the total collagen produced, the decrease in overall collagen expression reflects a specific suppression by EGF on type I homotrimer production in mesangial cells. As EGF exposure resulted in a decrease in collagen production, these results suggest that the increases in synthesis and deposition of collagen observed in several glomerular diseases likely do not reflect the short-term effects of EGF on mesangial cells. Rather, these findings suggest the possibility that EGF or EGF-like growth factors may ameliorate the effects of other soluble factors that cause enhanced matrix production and deposition in renal diseases.     

NG2 proteoglycan increases mesangial cell proliferation and extracellular matrix production

As a membrane-spanning protein, NG2 chondroitin sulfate proteoglycan interacts with molecules on both sides of plasma membrane. The present study explored the role of NG2 in the pathogenesis of diabetic nephropathy. In the normal kidneys, NG2 was observed predominantly in glomerular mesangium, Bowman's capsule and interstitial vessels. Both mRNA and protein expression in kidneys was significantly higher in strepozotocin-induced diabetic rats than that in normal rats. In the cultured rat mesangial cell line HBZY-1, overexpression of NG2 promoted mesangial cell proliferation and extracellular matrix (ECM) production, such as type VI collagen and laminin. Furthermore, target knockdown of NG2 resulted in decreased cell proliferation and ECM formation. The observations suggest that NG2 is up-regulated in diabetic nephropathy. It actively participates in the development and progression of glomerulosclerosis by stimulating proliferation of mesangial cells and deposition of ECM.     

Corticotropin-releasing hormone induces proliferation and TNF-alpha release in cultured rat microglia via MAP kinase signalling pathways

We have previously demonstrated that corticotropin-releasing hormone (CRH) receptor 1 (CRH-R1) is functionally expressed in rat microglia. In the present study, we show that CRH, acting on CRH-R1, promoted cell proliferation and tumour necrosis factor-alpha (TNF-alpha) release in cultured rat microglia. Exogenous CRH resulted in an increase in BrdU incorporation compared with control cells, which was observed in a range of concentrations of CRH between 10 and 500 nm, with a maximal response at 50 nm. The effect of CRH on BrdU incorporation was inhibited by a CRH antagonist astressin but not by a cAMP-dependent protein kinase inhibitor H89. Exposure of microglial cells to CRH resulted in a transient and rapid increase in TNF-alpha release in a dose-dependent manner. In the presence of astressin, the effects of CRH on TNF-alpha release were attenuated. CRH effects on TNF-alpha release were also inhibited by specific inhibitors of MEK, the upstream kinase of the extracellular signal-regulated protein kinase (ERK) (PD98059) or p38 mitogen-activated protein kinase (SB203580), but not by H89. Furthermore, CRH induced rapid phosphorylation of ERK and p38 kinases. Astressin, PD98059, and SB230580 were able to inhibit CRH-induced kinase phosphorylation. These results suggest that CRH induces cell proliferation and TNF-alpha release in cultured microglia via MAP kinase signalling pathways, thereby providing insight into the interactions between CRH and inflammatory mediators.     

Attenuation of peroxisome proliferator-activated receptor gamma (PPARgamma) mediates gastrin-stimulated colorectal cancer cell proliferation

Peroxisome proliferators-activated receptor gamma (PPARgamma) has been shown to suppress cell proliferation and tumorigenesis, whereas the gastrointestinal regulatory peptide gastrin stimulates the growth of neoplastic cells. The present studies were directed to determine whether changes in PPARgamma expression might mediate the effects of gastrin on the proliferation of colorectal cancer (CRC). Initially, using growth assays, we determined that the human CRC cell line DLD-1 expressed both functional PPARgamma and gastrin receptors. Amidated gastrin (G-17) attenuated the growth suppressing effects of PPARgamma by decreasing PPARgamma activity and total protein expression, in part through an increase in the rate of proteasomal degradation. G-17-induced degradation of PPARgamma appeared to be mediated through phosphorylation of PPARgamma at serine 84 by a process involving the biphasic phosphorylation of ERK1/2 and activation of the epidermal growth factor receptor (EGFR). These results were confirmed through the use of EGFR antagonist AG1478 and MEK1 inhibitor PD98059. Furthermore, mutation of PPARgamma at serine 84 reduced the effects of G-17, as evident by inability of G-17 to attenuate PPARgamma promoter activity, degrade PPARgamma, or inhibit the growth suppressing effects of PPARgamma. The results of these studies demonstrate that the trophic properties of gastrin in CRC may be mediated in part by transactivation of the EGFR and phosphorylation of ERK1/2, leading to degradation of PPARgamma protein and a decrease in PPARgamma activation.     

Retracted: Effects of microRNA-370 on mesangial cell proliferation and extracellular matrix accumulation by binding to canopy 1 in a rat model of diabetic nephropathy

As one major diabetic complication, diabetic nephropathy (DN) has been reported to be associated with various kinds of microRNA (miRNA). Thus, we conducted this study to explore the potential of miR-370 in a rat model of DN through investigation of mesangial cell proliferation and extracellular matrix (ECM). A total of 40 healthy adult male Sprague–Dawley rats were enrolled and assigned into normal (n = 10) and DN ( n = 30, DN rat model) groups. Dual-luciferase reporter assay was performed for the targeting relationship between miR-370 and canopy 1 (CNPY1). Mesangial cells were collected and transfected with prepared mimic, inhibitor or small interfering RNA (siRNA) for analyzing the effect of miR-370 on DN mice with the help of expression and cell biological processes detection. CNPY1 was confirmed as a target gene of miR-370. DN mice had increased expression of miR-370, fibronectin, type I collagen (Col I), type IV collagen (Col IV), and plasminogen activator inhibitor-1 (PAI-1) but reduced CNPY1 expression. Cells transfected with miR-370 mimic and siRNA–CNPY1 had increased expression of fibronectin, Col I, Col IV, and PAI-1 but decreased CNPY1 expression. The miR-370 mimic and siRNA–CNPY1 groups showed increased cell proliferation, as well as elevated ECM accumulation and declined cell apoptosis rate as compared with the blank and negative control groups, with reverse trends observed in the miR-370 inhibitor group. Our study concludes that overexpression of miR-370 promotes mesangial cell proliferation and ECM accumulation by suppressing CNPY1 in a rat model of DN.     

PI3K-AKT pathway mediates growth and survival signals during development of fetal mouse lung

We examined the roles of the PI3K-AKT signalling pathway in fetal lung development. By Western blotting, phosphorylated AKT (pAKT) was highly expressed in fetal days 12 and 14 with decreased expression thereafter. By immunohistochemistry, pAKT was expressed mainly in the respiratory epithelium of early fetal days. We examined the effects of fibroblast growth factor 1 (FGF1), PI3K inhibitors (LY294002 and wortmannin), MAPK inhibitor (PD98059) and both of FGF1 and each inhibitor on lung morphogenesis, BrdU incorporation and apoptosis. In the FGF1-treated explants, the number of terminal buds and BrdU-labelled cells increased significantly, while the LY294002-, wortmannin-, PD98059-treated explants demonstrated obvious decreases. The effects by FGF1 were inhibited by LY294002, wortmannin and PD98059. Regardless of the presence of FGF1, the LY294002-, wortmannin- and PD98059-treated explants increased apoptosis revealed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling assay in the mesenchyme of the explants. At the same time, the effect of LY294002, wortmannin, PD98059 on expression of surfactant apoprotein C (SPC) were also studied. The LY294002 and wortmannin treatments showed decreased expression of SPC. These findings suggest that the PI3K-AKT signalling pathway plays a pivotal role in mouse lung development through various biological processes.