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Smad signaling and extracellular matrix proteins upregulation in rat mesangial cells
Biochemical and Biophysical Research Communications xxx (2016) 1e6
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Parathyroid hormone inhibits TGF-b/Smad signaling and extracellular matrix proteins upregulation in rat mesangial cells Fang-Fang Peng, Ze-Ling Xiao, Hong-Min Chen, Yan Chen, Jian Zhou, Hong Yu, Bai-Fang Zhang* Department of Biochemistry and Hubei Provincial Key Laboratory of Developmentally Originated Disease, Wuhan University School of Basic Medical Sciences, Wuhan, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 July 2016 Accepted 11 August 2016 Available online xxx
Accumulation of glomerular matrix is a hallmark of diabetic nephropathy. TGF-b1 is a major cytokine mediating the production of various extracellular matrix (ECM) proteins. The aim of this study is to elucidate the effect of parathyroid hormone (PTH) on TGF-b1 and high glucose-induced upregulation of ECM proteins in primary mesangial cells from Sprague-Dawley rat. The results showed that PTH pretreatment prevented TGF-b1 and high glucose-induced Smad2/3 phosphorylation and consequent upregulation of fibronectin and type IV collagen within 4 h. The inhibitory effect of PTH is due to PTH1R activation, because knocking down PTH 1 receptor (PTH1R) by RNA interference reversed the inhibitory effect of PTH on TGF-b1 and high glucose-induced Smad2/3 phosphorylation and ECM upregulation. Furthermore, it is found that PTH1R associated with TGF-b type II receptor (TbR II) and both receptors internalized into the cytoplasm when mesangial cells were stimulated with PTH alone. The internalization of TbR II might reduce the amount of membrane TbR II, attenuate the sensitivity of mesangial cells to TGF-b1, and therefore inhibit Smad activation and ECM upregulation induced by TGF-b1 and high glucose. Further studies are needed to know whether the endocytic receptors are to be degraded or recycled, and evaluate the role of PTH in TGF-b1 signaling more comprehensively. © 2016 Elsevier Inc. All rights reserved.
Keywords: Extracellular matrix Mesangial cells Parathyroid hormone TGF-b
1. Introduction Parathyroid hormone (PTH) is a polypeptide hormone containing 84 amino acids, synthesized and secreted by the main cells in the parathyroid gland [1]. PTH can regulate plasma calcium and phosphorus concentrations by acting on the specific receptors mainly distributed in bone and kidney [2]. There are two types of high-affinity receptors for PTH: PTH 1 receptor (PTH1R) and PTH 2 receptor (PTH2R). PTH1R is highly expressed in bone and kidney [3,4], and PTH2R is mainly located on the central nervous system, pancreas, testis and placenta [5]. In the kidney PTH promotes calcium reabsorption, stimulates phosphate excretion and increase 1alpha-hydroxylase activity to active vitamin D by binding to PTH1R [6]. In addition, PTH affects glomerular filtration rate probably by modulating the action of podocytes [7,8]. It has been reported that PTH1R is also expressed in mesangial cells [9]. PTH1R activation by
* Corresponding author. Department of Biochemistry, Wuhan University School of Basic Medical Sciences, 185 Dong Hu Road, Wuhan, Hubei 430071, PR China. E-mail address: [email protected] (B.-F. Zhang).
PTH (1e34) had no effect on proliferation but improved mesangial cell survival [10]. Transformation growth factor-beta (TGF-b) and its specific membrane receptors including TGF-b type I receptor (TbR I) and TbR II are widely expressed in a variety of tissues including kidney. TGF-b induces TbR I and TbR II binding, then TbR II enables TbR I phosphorylation, resulting in the activation of TbR I, the phosphorylation of Smad2/3 protein or other signal molecules, triggering downstream pathways [11]. TGF-b has been considered an important mediator in the pathogenesis of chronic kidney diseases characterized by extracellular matrix (ECM) accumulation, glomerulosclerosis and tubulointerstitial fibrosis [12e14]. Mesangial cells are the intrinsic cells of kidney and crucial for glomerular function [15,16]. Strong evidence has been shown that excessive deposition of ECM proteins in the mesangium is a major event in the glomerulosclerosis of several kidney diseases including diabetic nephropathy [17]. TGF-b plays an important role in mesangium expansion and consequent ECM accumulation. TGF-b can induce the production of different ECM proteins such as fibronectin, type I and IV collagen, laminin and heparan sulfate proteoglycan in cultured mesangial cells [18e21]. Many studies
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Please cite this article in press as: F.-F. Peng, et al., Parathyroid hormone inhibits TGF-b/Smad signaling and extracellular matrix proteins upregulation in rat mesangial cells, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.08.073
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Fig. 1. PTH pretreatment prevented TGF-b1 and HG-induced phosphorylation of Smad2 and Smad3 and upregulation of ECM protein. Primary rat mesangial cells were incubated with 100 nM PTH for 30 min followed by TGF-b1 or HG treatment for indicated time. A, C: Smad2 and Smad3 phosphorylation were assessed by Western blot (*P < 0.05 vs. control, #P < 0.05 vs. HG group, n ¼ 5). B, D: The protein levels of fibronectin and collagen IV were assessed by Western blot (*P < 0.05 vs. control, #P < 0.05 vs. TGF-b1 or HG group, n ¼ 6).
have illuminated the interactions of TGF-b and PTH signaling pathways in bone [22e24], however, little is known about the crosstalk between TGF-b and PTH in kidney. The present study is to investigate whether PTH has an effect on high glucose (HG) and TGF-b1-induced ECM upregulation in rat mesangial cells. 2. Materials and methods 2.1. Cell culture and treatments Primary mesangial cells obtained from Sprague-Dawley rats (animal center, Wuhan University, China) were cultured in DMEM with 20% fetal calf serum (Invitrogen, Carlsbad, USA), containing 5.6 mM glucose, 100 mg/ml streptomycin and 100 units/ml penicillin. Confluent mesangial cells were made quiescent by incubation for 24 h in serum-free medium prior to the following treatment. Human PTH (1e34) (hereafter referred to as PTH) (Bachem, Bubendorf, Swiss) was used at 100 nM, human TGF-b1 (R&D Systems, Minneapolis, USA) was used at 2 ng/ml, D-glucose was added at 24.4 mM for high glucose levels (final concentration 30 mM), and mannitol (24.4 mM) was added for osmotic control. 2.2. Protein extraction and Western blotting For isolating total cellular protein, cells were harvested in regular lysis buffer and cell lysates were centrifuged at 4 C for 10 min
to collect supernatant. For isolating cytosolic fractions and membrane fractions, cells were first lysed with hypotonic lysis buffer and then lysed with regular lysis buffer including 60 mM N-octylglucopyranoside as recently described [25]. After protein concentration was determined, 50 mg of total protein was separated on SDS-PAGE followed by Western blot. Primary antibodies included monoclonal TbR II antibody (1:1000, Santa Cruz Biotechnology, Santa Cruz, USA), polyclonal PTH1R antibody PRB-640P (1:1000, Covance, Princeton, USA), polyclonal phospho-Smad2 antibody S465/467 (1:1000), polyclonal phospho-Smad3 antibody S433/435 (1:1000), polyclonal Smad2/3 antibody (1:1000, all Cell Signaling, Danvers, USA), polyclonal type IV Collagen antibody (1:1000, Santa Cruz), monoclonal fibronectin antibody (1:2000, BD Biosciences, San Jose, USA), and monoclonal b-actin antibody (1:5000, Sigma, St. Louis, USA). 2.3. Immunoprecipitation Cells were lysed with IP lysis buffer containing 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.5% Triton X-100, 10% glycerol, 5 mM EDTA, 1 mM DTT, 60 mM N-octyl-glucopyranoside, and protease inhibitors. Equal amounts of lysate were immunoprecipitated with different primary antibodies (2 mg) overnight rotating at 4 C. Protein G-agarose (30 ml) was then added to absorb antibodies for 90 min at 4 C. The immunoprecipitates were separated on SDSPAGE, and then Western blot was performed.
Please cite this article in press as: F.-F. Peng, et al., Parathyroid hormone inhibits TGF-b/Smad signaling and extracellular matrix proteins upregulation in rat mesangial cells, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.08.073
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Fig. 2. PTH1R RNAi reversed the blocking effect of PTH on TGF-b1-induced Smad2/3 phosphorylation and ECM upregulation. After transfection with PTH1R siRNA, rat mesangial cells were pretreated with PTH for 30 min before TGF-b1 treatment for indicated duration. A: PTH1R downregulation was detected by Western blot. B: Smad2 and Smad3 phosphorylation were assessed by Western blot (*P < 0.05 vs. control, n ¼ 6). C: The protein levels of fibronectin and collagen IV were assessed by Western blot (*P < 0.05 vs. control, # P < 0.05 vs. TGF-b1 group, n ¼ 4).
2.4. RNA interference
3. Results
50e60% confluent mesangial cells were transfected with 100 nM PTH1R siRNA or control non-targeting siRNA (RiboBio, China). The upstream sequence of PTH1R siRNA is 50 -GGACGAUGUCUUUACCAAAdTdT-30 , and the downstream sequence is 50 UUUGGUAAAGACAUCGUCCdTdT-30 . Transfection was performed using Effectene kit (Qiagen, Germany) according to the manufacturer's instruction. After transfection for 18e24 h, mesangial cells were made quiescent with 0% serum for 24 h followed by the treatment with different reagents. Then cell lysates were collected as described above.
3.1. PTH pretreatment prevented TGF-b1 and HG-induced Smad2/3 activation and ECM upregulation
2.5. Statistical analysis All data are shown as mean ± SEM. Results were analyzed by one-way ANOVA with Turkey Honestly Significant Difference (HSD) for post hoc analysis (SPSS 17.0 for Windows). Statistical significance is P < 0.05. The number of independent experiments is given in figure legends (n).
TGF-b1 is the key cytokine mediating glomerular ECM accumulation by mesangial cells in chronic kidney diseases including diabetic nephropathy [26e28]. We first observed whether PTH regulates TGF-b1 signaling in rat mesangial cells. As shown in Fig. 1A, PTH pretreatment blocked TGF-b1-induced phosphorylation of Smad2 and Smad3. TGF-b1-induced upregulation of fibronectin and type IV collagen were also inhibited by PTH pretreatment (Fig. 1B). These results indicate that PTH attenuates TGF-b1/Smad signaling and ECM upregulation in mesangial cells. There is a lot of evidence showing that HG induces TGF-b1 upregulation and secretion in mesangial cells [21,29]. TGF-b1 is an important mediator of HG-induced fibrogenic responses [30e32]. We thus assessed the role of PTH in the context of HG stimulation. As illustrated in Fig. 1C, the phosphorylation of Smad2 and Smad3 increased in response to HG at 2 h, which was blocked by PTH pretreatment. HG-induced upregulation of fibronectin and type IV collagen were also inhibited by PTH pretreatment (Fig. 1D).
Please cite this article in press as: F.-F. Peng, et al., Parathyroid hormone inhibits TGF-b/Smad signaling and extracellular matrix proteins upregulation in rat mesangial cells, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.08.073
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Fig. 3. PTH1R RNAi relieved the inhibitory effect of PTH on HG-induced Smad2/3 phosphorylation and ECM upregulation. After transfection with PTH1R siRNA, cells were pretreated with PTH before HG incubation for indicated time. A: Smad2 and Smad3 phosphorylation were assessed by Western blot (*P < 0.05 vs. control, #P < 0.05 vs. HG group, n ¼ 5). B: The protein levels of fibronectin and collagen IV were assessed by Western blot (*P < 0.05 vs. control, #P < 0.05 vs. HG group, n ¼ 4).
3.2. PTH1R siRNA relieved the inhibitory effect of PTH on TGF-b1 and HG signaling in mesangial cells To elucidate the mechanism of the inhibitory effect of PTH on TGF-b1 and HG-induced ECM upregulation, we examined the role of PTH1R using RNA interference to knock down PTH1R. PTH1R protein level was successfully down-regulated in rat mesangial cells (Fig. 2A). PTH could not prevent TGF-b1-induced Smad2/3 phosphorylation and upregulation of fibronectin and type IV collagen when PTH1R was knocked down (Fig. 2B, C). This suggests that PTH1R plays an important role in PTH pretreatment-mediated the inhibition of TGF-b1 signaling. Knocking down PTH1R also relieved the inhibitory effect of PTH on HG-induced Smad2/3 phosphorylation and ECM upregulation (Fig. 3A, B), further confirming the importance of PTH1R. 3.3. PTH induced endocytosis of the PTH1R-TbR II complex in mesangial cells PTH1R activation induced by PTH pretreatment involved in TGF-
b1 signaling, suggesting that there is a crosstalk between PTH and TGF-b1 signaling. To assess this possibility, we sought a physical association between PTH1R and TbR I, TbR II in rat mesangial cells. PTH1R protein was only detected in anti-TbR II immunoprecipitates after PTH treatment, and vice versa (Fig. 4A, B). However, TbR II could not interact with PTH1R in response to TGF-b1 in immunoprecipitation assay (data not shown). These results suggest that PTH alone induces the formation of PTH1R-TbR II complex,
consistent with previous study in osteoblasts [33]. We next observed whether the formation PTH1R-TbR II complex lead to the internalization of both receptors into the cytoplasm. As seen in Fig. 4C and D, the internalization of TbR II was induced by PTH in a time-dependent fashion, similar to that of PTH1R. The amount of cell-surface TbR II and PTH1R decreased remarkably from 10 min to 30 min, while the amount of cytosol TbR II and PTH1R increased at the same time. Taken together, these data suggested that PTH alone induces the association of PTH1R with TbR II and the endocytic internalization of PTH1R and TbR II in rat mesangial cells.
4. Discussion PTH is one of the most important hormones that could regulate calcium and phosphorus metabolism and bone transformation [2,6]. The level of serum PTH increases significantly at the early stage of chronic renal failure. The main consequences of increased PTH levels are the development of renal osteopathy and cardiovascular damage [34e36]. However, there has very little direct experimental evidence on the renal effects of PTH, especially by acting on mesangial cells. Here we explored the effects of PTH on TGF-b1 and HG signaling in primary rat mesangial cells. Our data show that PTH inhibits the upregulation of fibronectin and type IV collagen induced by TGF-b1 and HG, and to the best of our knowledge, it has not been addressed previously. We first observed whether PTH has an impact on the main signaling pathway of TGF-b1, Smad2/3 pathway. Pretreatment with PTH prevented TGF-b1-induced Smad2 and Smad3 phosphoryla-
Please cite this article in press as: F.-F. Peng, et al., Parathyroid hormone inhibits TGF-b/Smad signaling and extracellular matrix proteins upregulation in rat mesangial cells, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.08.073
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Fig. 4. PTH1R associated with TbR II and both receptors internalized into the cytoplasm when mesangial cells were stimulated with PTH. Cells were incubated with 100 nM PTH for the indicated duration. A: Immunoprecipitation was performed using TbR II antibody, and PTH1R protein level was assessed by Western blot. B: Immunoprecipitation was carried out using PTH1R antibody and TbR II protein level was assessed by Western blot (*P < 0.05 vs. control, n ¼ 5). C, D: The protein levels of PTH1R and TbR II in the cell membrane and cytoplasm were detected by Western blot, respectively (*P < 0.05 vs. control, n ¼ 4).
tion, and thereby inhibited the upregulation of ECM proteins, fibronectin and type IV collagen. Accumulation of glomerular matrix is a hallmark of diabetic nephropathy [17,37]. Numerous studies have shown that high glucose induces TGF-b1 expression in cultured mesangial cells as well as in diabetic kidney [30,31,38,39]. TGF-b1 is a major cytokine mediating the production of various ECM proteins in mesangial cells [30e32]. We thus examined whether PTH could block HG-induced activation of TGF-b1 signaling. Our results showed that PTH also prevented Smad2/3 phosphorylation and upregulation of fibronectin and type IV collagen mediated by HG. The activation of PTH1R appears to be particularly important because PTH1R siRNA reversed the inhibitory effect of PTH on TGF-b1 and HG-induced ECM upregulation. We next investigated the interactions of PTH and TGF-b1 receptors. Our results show that PTH, but not TGF-b1, induced the formation of PTH1R-TbR II complex and subsequent endocytosis of both receptors in rat mesangial cells. This is in accordance with results reported previously by Qiu et al. They found that TbR II directly phosphorylated the cytoplasmic domain of PTH1R and facilitated PTH-induced endocytosis of the PTH1R-TbR II complex, which lead to the loss of sensitization of both TGF-b and PTH in
osteoblasts [33]. Our results further confirm that the same mechanism exists in mesangial cells. The internalization of TbR II induced by PTH might reduce the amount of membrane TbR II, attenuate the sensitivity of mesangial cells to TGF-b1, and therefore inhibit Smad activation and ECM upregulation induced by TGF-b1 and HG. PTH1R siRNA retains cell-surface TbR II, and therefore fail to inhibit TGF-b1/Smad signaling. Put together, these results indicate that PTH may inhibit TGF-b1 and HG-induced fibrogenic responses through PTH1R-dependent endocytosis of TbR II in mesangial cells. PTH induced internalization of PTH1R and TbR II, but the fate of the endocytic receptors is unknown. They might follow two classical intracellular pathways: degradation, or probably back to the cell membrane. Degradation of receptors will result in attenuation of sensitization to both TGF-b and PTH signaling, while recycling of receptors will restore cellular responses. If the receptor recycling occurs, PTH will lose its inhibitory effect on TGFb signaling. In this study, the duration time of treatment is no more than 4 h, and the internalized receptors might still wait for sorting in the cytoplasm. Additional experiments are required to elucidate this point. In conclusion, the present study reveals the crosstalk between
Please cite this article in press as: F.-F. Peng, et al., Parathyroid hormone inhibits TGF-b/Smad signaling and extracellular matrix proteins upregulation in rat mesangial cells, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.08.073
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PTH and TGF-b signaling in primary rat mesangial cells. The interaction between PTH1R and TbR II plays a essential role in the signal pathways. PTH induces the formation and internalization of PTH1RTbR II complex, and down-regulates TbR II in cell surface. Therefore, TGF-b1 and HG-induced activation of Smad2 or Smad3 and upregulation of ECM proteins are blocked. Thus, PTH seems to be a potential actor in fibrogenesis mediated by mesangial cells. Acknowledgement This work was supported by the National Natural Science Foundation of China [grant numbers 81370819 and 81070643]. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.bbrc.2016.08.073. Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2016.08.073. References [1] J.S. Rodman, H.O. Heinemann, Parathyroid hormone and the regulation of acid-base balance, Am. J. Med. Sci. 270 (1975) 481e489. [2] K.E. Poole, J. Reeve, Parathyroid hormone-a bone anabolic and catabolic agent, Curr. Opin. Pharmacol. 5 (2005) 612e617. ~ a, N. Ardaillou, R. Ardaillou, J. Deeds, G.V. Segre, [3] K. Lee, D. Brown, P. Uren Localization of parathyroid hormone/parathyroid hormone-related peptide receptor mRNA in kidney, Am. J. Physiol. 270 (1996) F186eF191. [4] M. Mannstadt, H. Jüppner, T.J. Gardella, Receptors for PTH and PTHrP: their biological importance and functional properties, Am. J. Physiol. 277 (1999) F665eF675. [5] T.B. Usdin, T.I. Bonner, S.R. Hoare, The parathyroid hormone 2 (PTH2) receptor, Recept. Channels 8 (2002) 211e218. [6] S.J. Khundmiri, R.D. Murray, E. Lederer, PTH and vitamin D, Compr. Physiol. 6 (2016) 561e601. [7] I. Ichikawa, H.D. Humes, T.P. Dousa, B.M. Brenner, Influence of parathyroid hormone on glomerular ultrafiltration in the rat, Am. J. Physiol. 234 (1978) F393eF401. [8] G.R. Marchand, Effect of parathyroid hormone on the determinants of glomerular filtration in dogs, Am. J. Physiol. 248 (1985) F482eF486. pez-Luna, A. Ortega, M. Romero, M.A. Guitie rrez-Tarre s, [9] A. Izquierdo, P. Lo I. Arribas, M.J. Alvarez, P. Esbrit, R.J. Bosch, The parathyroid hormone-related protein system and diabetic nephropathy outcome in streptozotocin-induced diabetes, Kidney Int. 69 (2006) 2171e2177. [10] M. Hochane, D. Raison, C. Coquard, O. Imhoff, T. Massfelder, B. Moulin, J.J. Helwig, M. Barthelmebs, Parathyroid hormone-related protein is a mitogenic and a survival factor of mesangial cells from male mice: role of intracrine and paracrine pathways, Endocrinology 154 (2013) 853e864. [11] E. Pardali, M.J. Goumans, P. ten Dijke, Signaling by members of the TGF-beta family in vascular morphogenesis and disease, Trends Cell Biol. 20 (2010) 556e567. €ttinger, TGF-beta in renal injury and disease, Semin. Nephrol. 27 (2007) [12] E.P. Bo 309e320. pez-Hern pez-Novoa, Role of TGF-b in chronic kidney [13] F.J. Lo andez, J.M. Lo disease: an integration of tubular, glomerular and vascular effects, Cell Tissue Res. 347 (2012) 141e154. [14] W. Wang, V. Koka, H.Y. Lan, Transforming growth factor-beta and Smad signalling in kidney diseases, Nephrology 10 (2005) 48e56. €ndorff, B. Banas, The mesangial cell revisited: no cell is an island, [15] D. Schlo
J. Am. Soc. Nephrol. 20 (2009) 1179e1187. [16] I. Sinuani, I. Beberashvili, Z. Averbukh, M. Cohn, I. Gitelman, J. Weissgarten, Mesangial cells initiate compensatory tubular cell hypertrophy, Am. J. Nephrol. 31 (2010) 326e331. [17] R.M. Mason, N.A. Wahab, Extracellular matrix metabolism in diabetic nephropathy, J. Am. Soc. Nephrol. 14 (2003) 1358e1373. [18] N.G. McKay, T.F. Khong, N.E. Haites, D.A. Power, The effect of transforming growth factor-1 on mesangial cell fibronectin synthesis: increased incorporation into the extracellular matrix and reduced pI but no effect on alternative splicing, Exp. Mol. Pathol. 59 (1993) 211e224. [19] A.C. Poncelet, H.W. Schnaper, Regulation of mesangial cell collagen turnover by transforming growth factor-beta1, Am. J. Physiol. Ren. Physiol. 275 (1998) F458eF466. [20] S. Suzuki, I. Ebihara, Y. Tomino, H. Koide, Transcriptional activation of matrix genes by transforming growth factor beta1 in mesangial cells, Exp. Nephrol. 1 (1993) 229e237. [21] V. Kolm, U. Sauer, B. Olgemooller, E.D. Schleicher, High glucose-induced TGFb1 regulates mesangial production of heparan sulfate proteoglycan, Am. J. Physiol. 270 (1996) F812eF821. [22] J.L. Crane, X. Cao, Bone marrow mesenchymal stem cells and TGF-b signaling in bone remodeling, J. Clin. Investig. 124 (2014) 466e472. [23] T. Tobimatsu, H. Kaji, H. Sowa, J. Naito, L. Canaff, G.N. Hendy, T. Sugimoto, K. Chihara, Parathyroid hormone increases beta-catenin levels through Smad3 in mouse osteoblastic cells, Endocrinology 147 (2006) 2583e2590. [24] Y. Wu, R. Kumar, Parathyroid hormone regulates transforming growth factor beta1 and beta2 synthesis in osteoblasts via divergent signaling pathways, J. Bone Miner. Res. 15 (2000) 879e884. [25] D. Wu, F. Peng, B. Zhang, A.J. Ingram, D.J. Kelly, R.E. Gilbert, B. Gao, S. Kumar, J.C. Krepinsky, PKC-beta1 mediates glucose-induced Akt activation and TGFbeta1 upregulation in mesangial cells, J. Am. Soc. Nephrol. 20 (2009) 554e566. [26] M. Isono, S. Chen, S.W. Hong, M.C. Iglesias-de la Cruz, F.N. Ziyadeh, Smad pathway is activated in the diabetic mouse kidney and Smad3 mediates TGFb-induced fibronectin in mesangial cells, Biochem. Biophys. Res. Commun. 296 (2002) 1356e1365. [27] H.S. Lee, C.Y. Song, Differential role of mesangial cells and podocytes in TGFbeta-induced mesangial matrix synthesis in chronic glomerular disease, Histol. Histopathol. 24 (2009) 901e908. [28] B.S. Weston, N.A. Wahab, R.M. Mason, CTGF mediates TGFb-induced fibronectin matrix deposition by upregulating active a5b1 integrin in human mesangial cells, J. Am. Soc. Nephrol. 14 (2003) 601e610. [29] V. Kolm-Litty, U. Sauer, A. Nerlich, R. Lehmann, E.D. Schleicher, High glucoseinduced transforming growth factor-b1 production is mediated by the hexosamine pathway in porcine glomerular mesangial cells, J. Clin. Investig. 101 (1998) 160e169. [30] J.H. Oh, H. Ha, M.R. Yu, H.B. Lee, Sequential effects of high glucose on mesangial cell transforming growth factor-b1 and fibronectin synthesis, Kidney Int. 54 (1998) 1872e1878. [31] N.A. Wahab, K. Harper, R.M. Mason, Expression of extracellular matrix molecules in human mesangial cells in response to prolonged hyperglycaemia, Biochem. J. 316 (1996) 985e992. [32] F.N. Zizadeh, K. Sharma, M. Ericksen, G. Wolf, Stimulation of collagen gene expression and protein synthesis in murine mesangial cells by high glucose is mediated by activation of transforming growth factor-b, J. Clin. Investig. 93 (1994) 536e542. [33] T. Qiu, X. Wu, F. Zhang, T.L. Clemens, M. Wan, X. Cao, TGF-beta type II receptor phosphorylates PTH receptor to integrate bone remodeling signaling, Nat. Cell Biol. 12 (2010) 224e234. [34] S.G. Rostand, T.B. Drueke, Parathyroid hormone, vitamin D, and cardiovascular disease in chronic renal failure, Kidney Int. 56 (1999) 383e392. [35] I.B. Salusky, W.G. Goodman, Adynamic renal osteodystrophy: is there a problem? J. Am. Soc. Nephrol. 12 (2001) 1978e1985. [36] E. Slatopolsky, K. Martin, K. Hruska, Parathyroid hormone metabolism and its potential as a uremic toxin, Am. J. Physiol. 239 (1980) F1eF12. [37] S.O. Kolset, F.P. Reinholt, T. Jenssen, Diabetic nephropathy and extracellular matrix, J. Histochem. Cytochem. 60 (2012) 976e986. [38] K. Sharma, F.N. Ziyadeh, Hyperglycemia and diabetic kidney disease. The case for transforming growth factor-b as a key mediator, Diabetes 44 (1995) 439e446. [39] E. Schleicher, A. Nerlich, The role of hyperglycemia in the development of diabetic complications, Horm. Metab. Res. 28 (1996) 367e373.
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Parathyroid hormone inhibits TGF-b/Smad signaling and extracellular matrix proteins upregulation in rat mesangial cells Fang-Fang Peng, Ze-Ling Xiao, Hong-Min Chen, Yan Chen, Jian Zhou, Hong Yu, Bai-Fang Zhang* Department of Biochemistry and Hubei Provincial Key Laboratory of Developmentally Originated Disease, Wuhan University School of Basic Medical Sciences, Wuhan, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 July 2016 Accepted 11 August 2016 Available online xxx
Accumulation of glomerular matrix is a hallmark of diabetic nephropathy. TGF-b1 is a major cytokine mediating the production of various extracellular matrix (ECM) proteins. The aim of this study is to elucidate the effect of parathyroid hormone (PTH) on TGF-b1 and high glucose-induced upregulation of ECM proteins in primary mesangial cells from Sprague-Dawley rat. The results showed that PTH pretreatment prevented TGF-b1 and high glucose-induced Smad2/3 phosphorylation and consequent upregulation of fibronectin and type IV collagen within 4 h. The inhibitory effect of PTH is due to PTH1R activation, because knocking down PTH 1 receptor (PTH1R) by RNA interference reversed the inhibitory effect of PTH on TGF-b1 and high glucose-induced Smad2/3 phosphorylation and ECM upregulation. Furthermore, it is found that PTH1R associated with TGF-b type II receptor (TbR II) and both receptors internalized into the cytoplasm when mesangial cells were stimulated with PTH alone. The internalization of TbR II might reduce the amount of membrane TbR II, attenuate the sensitivity of mesangial cells to TGF-b1, and therefore inhibit Smad activation and ECM upregulation induced by TGF-b1 and high glucose. Further studies are needed to know whether the endocytic receptors are to be degraded or recycled, and evaluate the role of PTH in TGF-b1 signaling more comprehensively. © 2016 Elsevier Inc. All rights reserved.
Keywords: Extracellular matrix Mesangial cells Parathyroid hormone TGF-b
1. Introduction Parathyroid hormone (PTH) is a polypeptide hormone containing 84 amino acids, synthesized and secreted by the main cells in the parathyroid gland [1]. PTH can regulate plasma calcium and phosphorus concentrations by acting on the specific receptors mainly distributed in bone and kidney [2]. There are two types of high-affinity receptors for PTH: PTH 1 receptor (PTH1R) and PTH 2 receptor (PTH2R). PTH1R is highly expressed in bone and kidney [3,4], and PTH2R is mainly located on the central nervous system, pancreas, testis and placenta [5]. In the kidney PTH promotes calcium reabsorption, stimulates phosphate excretion and increase 1alpha-hydroxylase activity to active vitamin D by binding to PTH1R [6]. In addition, PTH affects glomerular filtration rate probably by modulating the action of podocytes [7,8]. It has been reported that PTH1R is also expressed in mesangial cells [9]. PTH1R activation by
* Corresponding author. Department of Biochemistry, Wuhan University School of Basic Medical Sciences, 185 Dong Hu Road, Wuhan, Hubei 430071, PR China. E-mail address: [email protected] (B.-F. Zhang).
PTH (1e34) had no effect on proliferation but improved mesangial cell survival [10]. Transformation growth factor-beta (TGF-b) and its specific membrane receptors including TGF-b type I receptor (TbR I) and TbR II are widely expressed in a variety of tissues including kidney. TGF-b induces TbR I and TbR II binding, then TbR II enables TbR I phosphorylation, resulting in the activation of TbR I, the phosphorylation of Smad2/3 protein or other signal molecules, triggering downstream pathways [11]. TGF-b has been considered an important mediator in the pathogenesis of chronic kidney diseases characterized by extracellular matrix (ECM) accumulation, glomerulosclerosis and tubulointerstitial fibrosis [12e14]. Mesangial cells are the intrinsic cells of kidney and crucial for glomerular function [15,16]. Strong evidence has been shown that excessive deposition of ECM proteins in the mesangium is a major event in the glomerulosclerosis of several kidney diseases including diabetic nephropathy [17]. TGF-b plays an important role in mesangium expansion and consequent ECM accumulation. TGF-b can induce the production of different ECM proteins such as fibronectin, type I and IV collagen, laminin and heparan sulfate proteoglycan in cultured mesangial cells [18e21]. Many studies
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Please cite this article in press as: F.-F. Peng, et al., Parathyroid hormone inhibits TGF-b/Smad signaling and extracellular matrix proteins upregulation in rat mesangial cells, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.08.073
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Fig. 1. PTH pretreatment prevented TGF-b1 and HG-induced phosphorylation of Smad2 and Smad3 and upregulation of ECM protein. Primary rat mesangial cells were incubated with 100 nM PTH for 30 min followed by TGF-b1 or HG treatment for indicated time. A, C: Smad2 and Smad3 phosphorylation were assessed by Western blot (*P < 0.05 vs. control, #P < 0.05 vs. HG group, n ¼ 5). B, D: The protein levels of fibronectin and collagen IV were assessed by Western blot (*P < 0.05 vs. control, #P < 0.05 vs. TGF-b1 or HG group, n ¼ 6).
have illuminated the interactions of TGF-b and PTH signaling pathways in bone [22e24], however, little is known about the crosstalk between TGF-b and PTH in kidney. The present study is to investigate whether PTH has an effect on high glucose (HG) and TGF-b1-induced ECM upregulation in rat mesangial cells. 2. Materials and methods 2.1. Cell culture and treatments Primary mesangial cells obtained from Sprague-Dawley rats (animal center, Wuhan University, China) were cultured in DMEM with 20% fetal calf serum (Invitrogen, Carlsbad, USA), containing 5.6 mM glucose, 100 mg/ml streptomycin and 100 units/ml penicillin. Confluent mesangial cells were made quiescent by incubation for 24 h in serum-free medium prior to the following treatment. Human PTH (1e34) (hereafter referred to as PTH) (Bachem, Bubendorf, Swiss) was used at 100 nM, human TGF-b1 (R&D Systems, Minneapolis, USA) was used at 2 ng/ml, D-glucose was added at 24.4 mM for high glucose levels (final concentration 30 mM), and mannitol (24.4 mM) was added for osmotic control. 2.2. Protein extraction and Western blotting For isolating total cellular protein, cells were harvested in regular lysis buffer and cell lysates were centrifuged at 4 C for 10 min
to collect supernatant. For isolating cytosolic fractions and membrane fractions, cells were first lysed with hypotonic lysis buffer and then lysed with regular lysis buffer including 60 mM N-octylglucopyranoside as recently described [25]. After protein concentration was determined, 50 mg of total protein was separated on SDS-PAGE followed by Western blot. Primary antibodies included monoclonal TbR II antibody (1:1000, Santa Cruz Biotechnology, Santa Cruz, USA), polyclonal PTH1R antibody PRB-640P (1:1000, Covance, Princeton, USA), polyclonal phospho-Smad2 antibody S465/467 (1:1000), polyclonal phospho-Smad3 antibody S433/435 (1:1000), polyclonal Smad2/3 antibody (1:1000, all Cell Signaling, Danvers, USA), polyclonal type IV Collagen antibody (1:1000, Santa Cruz), monoclonal fibronectin antibody (1:2000, BD Biosciences, San Jose, USA), and monoclonal b-actin antibody (1:5000, Sigma, St. Louis, USA). 2.3. Immunoprecipitation Cells were lysed with IP lysis buffer containing 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.5% Triton X-100, 10% glycerol, 5 mM EDTA, 1 mM DTT, 60 mM N-octyl-glucopyranoside, and protease inhibitors. Equal amounts of lysate were immunoprecipitated with different primary antibodies (2 mg) overnight rotating at 4 C. Protein G-agarose (30 ml) was then added to absorb antibodies for 90 min at 4 C. The immunoprecipitates were separated on SDSPAGE, and then Western blot was performed.
Please cite this article in press as: F.-F. Peng, et al., Parathyroid hormone inhibits TGF-b/Smad signaling and extracellular matrix proteins upregulation in rat mesangial cells, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.08.073
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Fig. 2. PTH1R RNAi reversed the blocking effect of PTH on TGF-b1-induced Smad2/3 phosphorylation and ECM upregulation. After transfection with PTH1R siRNA, rat mesangial cells were pretreated with PTH for 30 min before TGF-b1 treatment for indicated duration. A: PTH1R downregulation was detected by Western blot. B: Smad2 and Smad3 phosphorylation were assessed by Western blot (*P < 0.05 vs. control, n ¼ 6). C: The protein levels of fibronectin and collagen IV were assessed by Western blot (*P < 0.05 vs. control, # P < 0.05 vs. TGF-b1 group, n ¼ 4).
2.4. RNA interference
3. Results
50e60% confluent mesangial cells were transfected with 100 nM PTH1R siRNA or control non-targeting siRNA (RiboBio, China). The upstream sequence of PTH1R siRNA is 50 -GGACGAUGUCUUUACCAAAdTdT-30 , and the downstream sequence is 50 UUUGGUAAAGACAUCGUCCdTdT-30 . Transfection was performed using Effectene kit (Qiagen, Germany) according to the manufacturer's instruction. After transfection for 18e24 h, mesangial cells were made quiescent with 0% serum for 24 h followed by the treatment with different reagents. Then cell lysates were collected as described above.
3.1. PTH pretreatment prevented TGF-b1 and HG-induced Smad2/3 activation and ECM upregulation
2.5. Statistical analysis All data are shown as mean ± SEM. Results were analyzed by one-way ANOVA with Turkey Honestly Significant Difference (HSD) for post hoc analysis (SPSS 17.0 for Windows). Statistical significance is P < 0.05. The number of independent experiments is given in figure legends (n).
TGF-b1 is the key cytokine mediating glomerular ECM accumulation by mesangial cells in chronic kidney diseases including diabetic nephropathy [26e28]. We first observed whether PTH regulates TGF-b1 signaling in rat mesangial cells. As shown in Fig. 1A, PTH pretreatment blocked TGF-b1-induced phosphorylation of Smad2 and Smad3. TGF-b1-induced upregulation of fibronectin and type IV collagen were also inhibited by PTH pretreatment (Fig. 1B). These results indicate that PTH attenuates TGF-b1/Smad signaling and ECM upregulation in mesangial cells. There is a lot of evidence showing that HG induces TGF-b1 upregulation and secretion in mesangial cells [21,29]. TGF-b1 is an important mediator of HG-induced fibrogenic responses [30e32]. We thus assessed the role of PTH in the context of HG stimulation. As illustrated in Fig. 1C, the phosphorylation of Smad2 and Smad3 increased in response to HG at 2 h, which was blocked by PTH pretreatment. HG-induced upregulation of fibronectin and type IV collagen were also inhibited by PTH pretreatment (Fig. 1D).
Please cite this article in press as: F.-F. Peng, et al., Parathyroid hormone inhibits TGF-b/Smad signaling and extracellular matrix proteins upregulation in rat mesangial cells, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.08.073
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Fig. 3. PTH1R RNAi relieved the inhibitory effect of PTH on HG-induced Smad2/3 phosphorylation and ECM upregulation. After transfection with PTH1R siRNA, cells were pretreated with PTH before HG incubation for indicated time. A: Smad2 and Smad3 phosphorylation were assessed by Western blot (*P < 0.05 vs. control, #P < 0.05 vs. HG group, n ¼ 5). B: The protein levels of fibronectin and collagen IV were assessed by Western blot (*P < 0.05 vs. control, #P < 0.05 vs. HG group, n ¼ 4).
3.2. PTH1R siRNA relieved the inhibitory effect of PTH on TGF-b1 and HG signaling in mesangial cells To elucidate the mechanism of the inhibitory effect of PTH on TGF-b1 and HG-induced ECM upregulation, we examined the role of PTH1R using RNA interference to knock down PTH1R. PTH1R protein level was successfully down-regulated in rat mesangial cells (Fig. 2A). PTH could not prevent TGF-b1-induced Smad2/3 phosphorylation and upregulation of fibronectin and type IV collagen when PTH1R was knocked down (Fig. 2B, C). This suggests that PTH1R plays an important role in PTH pretreatment-mediated the inhibition of TGF-b1 signaling. Knocking down PTH1R also relieved the inhibitory effect of PTH on HG-induced Smad2/3 phosphorylation and ECM upregulation (Fig. 3A, B), further confirming the importance of PTH1R. 3.3. PTH induced endocytosis of the PTH1R-TbR II complex in mesangial cells PTH1R activation induced by PTH pretreatment involved in TGF-
b1 signaling, suggesting that there is a crosstalk between PTH and TGF-b1 signaling. To assess this possibility, we sought a physical association between PTH1R and TbR I, TbR II in rat mesangial cells. PTH1R protein was only detected in anti-TbR II immunoprecipitates after PTH treatment, and vice versa (Fig. 4A, B). However, TbR II could not interact with PTH1R in response to TGF-b1 in immunoprecipitation assay (data not shown). These results suggest that PTH alone induces the formation of PTH1R-TbR II complex,
consistent with previous study in osteoblasts [33]. We next observed whether the formation PTH1R-TbR II complex lead to the internalization of both receptors into the cytoplasm. As seen in Fig. 4C and D, the internalization of TbR II was induced by PTH in a time-dependent fashion, similar to that of PTH1R. The amount of cell-surface TbR II and PTH1R decreased remarkably from 10 min to 30 min, while the amount of cytosol TbR II and PTH1R increased at the same time. Taken together, these data suggested that PTH alone induces the association of PTH1R with TbR II and the endocytic internalization of PTH1R and TbR II in rat mesangial cells.
4. Discussion PTH is one of the most important hormones that could regulate calcium and phosphorus metabolism and bone transformation [2,6]. The level of serum PTH increases significantly at the early stage of chronic renal failure. The main consequences of increased PTH levels are the development of renal osteopathy and cardiovascular damage [34e36]. However, there has very little direct experimental evidence on the renal effects of PTH, especially by acting on mesangial cells. Here we explored the effects of PTH on TGF-b1 and HG signaling in primary rat mesangial cells. Our data show that PTH inhibits the upregulation of fibronectin and type IV collagen induced by TGF-b1 and HG, and to the best of our knowledge, it has not been addressed previously. We first observed whether PTH has an impact on the main signaling pathway of TGF-b1, Smad2/3 pathway. Pretreatment with PTH prevented TGF-b1-induced Smad2 and Smad3 phosphoryla-
Please cite this article in press as: F.-F. Peng, et al., Parathyroid hormone inhibits TGF-b/Smad signaling and extracellular matrix proteins upregulation in rat mesangial cells, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.08.073
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Fig. 4. PTH1R associated with TbR II and both receptors internalized into the cytoplasm when mesangial cells were stimulated with PTH. Cells were incubated with 100 nM PTH for the indicated duration. A: Immunoprecipitation was performed using TbR II antibody, and PTH1R protein level was assessed by Western blot. B: Immunoprecipitation was carried out using PTH1R antibody and TbR II protein level was assessed by Western blot (*P < 0.05 vs. control, n ¼ 5). C, D: The protein levels of PTH1R and TbR II in the cell membrane and cytoplasm were detected by Western blot, respectively (*P < 0.05 vs. control, n ¼ 4).
tion, and thereby inhibited the upregulation of ECM proteins, fibronectin and type IV collagen. Accumulation of glomerular matrix is a hallmark of diabetic nephropathy [17,37]. Numerous studies have shown that high glucose induces TGF-b1 expression in cultured mesangial cells as well as in diabetic kidney [30,31,38,39]. TGF-b1 is a major cytokine mediating the production of various ECM proteins in mesangial cells [30e32]. We thus examined whether PTH could block HG-induced activation of TGF-b1 signaling. Our results showed that PTH also prevented Smad2/3 phosphorylation and upregulation of fibronectin and type IV collagen mediated by HG. The activation of PTH1R appears to be particularly important because PTH1R siRNA reversed the inhibitory effect of PTH on TGF-b1 and HG-induced ECM upregulation. We next investigated the interactions of PTH and TGF-b1 receptors. Our results show that PTH, but not TGF-b1, induced the formation of PTH1R-TbR II complex and subsequent endocytosis of both receptors in rat mesangial cells. This is in accordance with results reported previously by Qiu et al. They found that TbR II directly phosphorylated the cytoplasmic domain of PTH1R and facilitated PTH-induced endocytosis of the PTH1R-TbR II complex, which lead to the loss of sensitization of both TGF-b and PTH in
osteoblasts [33]. Our results further confirm that the same mechanism exists in mesangial cells. The internalization of TbR II induced by PTH might reduce the amount of membrane TbR II, attenuate the sensitivity of mesangial cells to TGF-b1, and therefore inhibit Smad activation and ECM upregulation induced by TGF-b1 and HG. PTH1R siRNA retains cell-surface TbR II, and therefore fail to inhibit TGF-b1/Smad signaling. Put together, these results indicate that PTH may inhibit TGF-b1 and HG-induced fibrogenic responses through PTH1R-dependent endocytosis of TbR II in mesangial cells. PTH induced internalization of PTH1R and TbR II, but the fate of the endocytic receptors is unknown. They might follow two classical intracellular pathways: degradation, or probably back to the cell membrane. Degradation of receptors will result in attenuation of sensitization to both TGF-b and PTH signaling, while recycling of receptors will restore cellular responses. If the receptor recycling occurs, PTH will lose its inhibitory effect on TGFb signaling. In this study, the duration time of treatment is no more than 4 h, and the internalized receptors might still wait for sorting in the cytoplasm. Additional experiments are required to elucidate this point. In conclusion, the present study reveals the crosstalk between
Please cite this article in press as: F.-F. Peng, et al., Parathyroid hormone inhibits TGF-b/Smad signaling and extracellular matrix proteins upregulation in rat mesangial cells, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.08.073
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PTH and TGF-b signaling in primary rat mesangial cells. The interaction between PTH1R and TbR II plays a essential role in the signal pathways. PTH induces the formation and internalization of PTH1RTbR II complex, and down-regulates TbR II in cell surface. Therefore, TGF-b1 and HG-induced activation of Smad2 or Smad3 and upregulation of ECM proteins are blocked. Thus, PTH seems to be a potential actor in fibrogenesis mediated by mesangial cells. Acknowledgement This work was supported by the National Natural Science Foundation of China [grant numbers 81370819 and 81070643]. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.bbrc.2016.08.073. Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2016.08.073. References [1] J.S. Rodman, H.O. Heinemann, Parathyroid hormone and the regulation of acid-base balance, Am. J. Med. Sci. 270 (1975) 481e489. [2] K.E. Poole, J. Reeve, Parathyroid hormone-a bone anabolic and catabolic agent, Curr. Opin. Pharmacol. 5 (2005) 612e617. ~ a, N. Ardaillou, R. Ardaillou, J. Deeds, G.V. Segre, [3] K. Lee, D. Brown, P. Uren Localization of parathyroid hormone/parathyroid hormone-related peptide receptor mRNA in kidney, Am. J. Physiol. 270 (1996) F186eF191. [4] M. Mannstadt, H. Jüppner, T.J. Gardella, Receptors for PTH and PTHrP: their biological importance and functional properties, Am. J. Physiol. 277 (1999) F665eF675. [5] T.B. Usdin, T.I. Bonner, S.R. Hoare, The parathyroid hormone 2 (PTH2) receptor, Recept. Channels 8 (2002) 211e218. [6] S.J. Khundmiri, R.D. Murray, E. Lederer, PTH and vitamin D, Compr. Physiol. 6 (2016) 561e601. [7] I. Ichikawa, H.D. Humes, T.P. Dousa, B.M. Brenner, Influence of parathyroid hormone on glomerular ultrafiltration in the rat, Am. J. Physiol. 234 (1978) F393eF401. [8] G.R. Marchand, Effect of parathyroid hormone on the determinants of glomerular filtration in dogs, Am. J. Physiol. 248 (1985) F482eF486. pez-Luna, A. Ortega, M. Romero, M.A. Guitie rrez-Tarre s, [9] A. Izquierdo, P. Lo I. Arribas, M.J. Alvarez, P. Esbrit, R.J. Bosch, The parathyroid hormone-related protein system and diabetic nephropathy outcome in streptozotocin-induced diabetes, Kidney Int. 69 (2006) 2171e2177. [10] M. Hochane, D. Raison, C. Coquard, O. Imhoff, T. Massfelder, B. Moulin, J.J. Helwig, M. Barthelmebs, Parathyroid hormone-related protein is a mitogenic and a survival factor of mesangial cells from male mice: role of intracrine and paracrine pathways, Endocrinology 154 (2013) 853e864. [11] E. Pardali, M.J. Goumans, P. ten Dijke, Signaling by members of the TGF-beta family in vascular morphogenesis and disease, Trends Cell Biol. 20 (2010) 556e567. €ttinger, TGF-beta in renal injury and disease, Semin. Nephrol. 27 (2007) [12] E.P. Bo 309e320. pez-Hern pez-Novoa, Role of TGF-b in chronic kidney [13] F.J. Lo andez, J.M. Lo disease: an integration of tubular, glomerular and vascular effects, Cell Tissue Res. 347 (2012) 141e154. [14] W. Wang, V. Koka, H.Y. Lan, Transforming growth factor-beta and Smad signalling in kidney diseases, Nephrology 10 (2005) 48e56. €ndorff, B. Banas, The mesangial cell revisited: no cell is an island, [15] D. Schlo
J. Am. Soc. Nephrol. 20 (2009) 1179e1187. [16] I. Sinuani, I. Beberashvili, Z. Averbukh, M. Cohn, I. Gitelman, J. Weissgarten, Mesangial cells initiate compensatory tubular cell hypertrophy, Am. J. Nephrol. 31 (2010) 326e331. [17] R.M. Mason, N.A. Wahab, Extracellular matrix metabolism in diabetic nephropathy, J. Am. Soc. Nephrol. 14 (2003) 1358e1373. [18] N.G. McKay, T.F. Khong, N.E. Haites, D.A. Power, The effect of transforming growth factor-1 on mesangial cell fibronectin synthesis: increased incorporation into the extracellular matrix and reduced pI but no effect on alternative splicing, Exp. Mol. Pathol. 59 (1993) 211e224. [19] A.C. Poncelet, H.W. Schnaper, Regulation of mesangial cell collagen turnover by transforming growth factor-beta1, Am. J. Physiol. Ren. Physiol. 275 (1998) F458eF466. [20] S. Suzuki, I. Ebihara, Y. Tomino, H. Koide, Transcriptional activation of matrix genes by transforming growth factor beta1 in mesangial cells, Exp. Nephrol. 1 (1993) 229e237. [21] V. Kolm, U. Sauer, B. Olgemooller, E.D. Schleicher, High glucose-induced TGFb1 regulates mesangial production of heparan sulfate proteoglycan, Am. J. Physiol. 270 (1996) F812eF821. [22] J.L. Crane, X. Cao, Bone marrow mesenchymal stem cells and TGF-b signaling in bone remodeling, J. Clin. Investig. 124 (2014) 466e472. [23] T. Tobimatsu, H. Kaji, H. Sowa, J. Naito, L. Canaff, G.N. Hendy, T. Sugimoto, K. Chihara, Parathyroid hormone increases beta-catenin levels through Smad3 in mouse osteoblastic cells, Endocrinology 147 (2006) 2583e2590. [24] Y. Wu, R. Kumar, Parathyroid hormone regulates transforming growth factor beta1 and beta2 synthesis in osteoblasts via divergent signaling pathways, J. Bone Miner. Res. 15 (2000) 879e884. [25] D. Wu, F. Peng, B. Zhang, A.J. Ingram, D.J. Kelly, R.E. Gilbert, B. Gao, S. Kumar, J.C. Krepinsky, PKC-beta1 mediates glucose-induced Akt activation and TGFbeta1 upregulation in mesangial cells, J. Am. Soc. Nephrol. 20 (2009) 554e566. [26] M. Isono, S. Chen, S.W. Hong, M.C. Iglesias-de la Cruz, F.N. Ziyadeh, Smad pathway is activated in the diabetic mouse kidney and Smad3 mediates TGFb-induced fibronectin in mesangial cells, Biochem. Biophys. Res. Commun. 296 (2002) 1356e1365. [27] H.S. Lee, C.Y. Song, Differential role of mesangial cells and podocytes in TGFbeta-induced mesangial matrix synthesis in chronic glomerular disease, Histol. Histopathol. 24 (2009) 901e908. [28] B.S. Weston, N.A. Wahab, R.M. Mason, CTGF mediates TGFb-induced fibronectin matrix deposition by upregulating active a5b1 integrin in human mesangial cells, J. Am. Soc. Nephrol. 14 (2003) 601e610. [29] V. Kolm-Litty, U. Sauer, A. Nerlich, R. Lehmann, E.D. Schleicher, High glucoseinduced transforming growth factor-b1 production is mediated by the hexosamine pathway in porcine glomerular mesangial cells, J. Clin. Investig. 101 (1998) 160e169. [30] J.H. Oh, H. Ha, M.R. Yu, H.B. Lee, Sequential effects of high glucose on mesangial cell transforming growth factor-b1 and fibronectin synthesis, Kidney Int. 54 (1998) 1872e1878. [31] N.A. Wahab, K. Harper, R.M. Mason, Expression of extracellular matrix molecules in human mesangial cells in response to prolonged hyperglycaemia, Biochem. J. 316 (1996) 985e992. [32] F.N. Zizadeh, K. Sharma, M. Ericksen, G. Wolf, Stimulation of collagen gene expression and protein synthesis in murine mesangial cells by high glucose is mediated by activation of transforming growth factor-b, J. Clin. Investig. 93 (1994) 536e542. [33] T. Qiu, X. Wu, F. Zhang, T.L. Clemens, M. Wan, X. Cao, TGF-beta type II receptor phosphorylates PTH receptor to integrate bone remodeling signaling, Nat. Cell Biol. 12 (2010) 224e234. [34] S.G. Rostand, T.B. Drueke, Parathyroid hormone, vitamin D, and cardiovascular disease in chronic renal failure, Kidney Int. 56 (1999) 383e392. [35] I.B. Salusky, W.G. Goodman, Adynamic renal osteodystrophy: is there a problem? J. Am. Soc. Nephrol. 12 (2001) 1978e1985. [36] E. Slatopolsky, K. Martin, K. Hruska, Parathyroid hormone metabolism and its potential as a uremic toxin, Am. J. Physiol. 239 (1980) F1eF12. [37] S.O. Kolset, F.P. Reinholt, T. Jenssen, Diabetic nephropathy and extracellular matrix, J. Histochem. Cytochem. 60 (2012) 976e986. [38] K. Sharma, F.N. Ziyadeh, Hyperglycemia and diabetic kidney disease. The case for transforming growth factor-b as a key mediator, Diabetes 44 (1995) 439e446. [39] E. Schleicher, A. Nerlich, The role of hyperglycemia in the development of diabetic complications, Horm. Metab. Res. 28 (1996) 367e373.
Please cite this article in press as: F.-F. Peng, et al., Parathyroid hormone inhibits TGF-b/Smad signaling and extracellular matrix proteins upregulation in rat mesangial cells, Biochemical and Biophysical Research Communications (2016), http://dx.doi.org/10.1016/j.bbrc.2016.08.073