Growth Hormone Induces Transforming Growth Factor‐Beta‐Induced Protein in Podocytes: Implications for Podocyte Depletion and Proteinuria

The glomerular podocytes form a major size selective barrier for the filtration of serum proteins and reduced podocyte number is a critical event in the pathogenesis of proteinuria during diabetic nephropathy (DN). An elevated level of growth hormone (GH) is implicated as a causative factor in the development of nephropathy in patients with type 1 diabetes mellitus. We have previously shown that podocytes express GH receptor and are a target for GH action. To elucidate the molecular basis for the effects of GH on podocyte depletion, we conducted PCR‐array analyses for extracellular matrix and adhesion molecules in podocytes. Our studies reveal that GH increases expression of a gene that encodes transforming growth factor‐beta‐induced protein (TGFBIp) expression. Similarly, microarray data retrieved from the Nephromine database revealed elevation of TGFBIp in patients with DN. Treatment with GH results in increased secretion of extracellular TGFBIp by podocytes. Both GH and TGFBIp induced apoptosis and epithelial mesenchymal transition (EMT) of podocytes. Exposure of podocytes to GH and TGFBIp resulted in increased migration of cells and altered podocyte permeability to albumin across podocyte monolayer. Administration of GH to rats induced EMT and apoptosis in the glomerular fraction of the kidney. Therefore, we conclude that the GH‐dependent increase in TGFBIp in the podocyte is one of the mechanisms responsible for podocyte depletion in DN. J. Cell. Biochem. 116: 1947–1956, 2015. © 2015 Wiley Periodicals, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/112213/1/jcb25150.pd

Proteomic Profiling of Mesenchymal Stem Cell Responses to Mechanical Strain and TGF-β1

Mesenchymal stem cells (MSCs) are a potential source of smooth muscle cells (SMCs) for constructing tissue-engineered vascular grafts. However, the details of how specific combinations of vascular microenvironmental factors regulate MSCs are not well understood. Previous studies have suggested that both mechanical stimulation with uniaxial cyclic strain and chemical stimulation with transforming growth factor-β1 (TGF-β1) can induce smooth muscle markers in MSCs. In this study, we investigated the combined effects of uniaxial cyclic strain and TGF-β1 stimulation on MSCs. By using a proteomic analysis, we found differential regulation of several proteins and genes, such as the up-regulation of TGF-β1-induced protein ig-h3 (BGH3) protein levels by TGF-β1 and up-regulation of calponin 3 protein level by cyclic strain. At the gene expression level, BGH3 was induced by TGF-β1, but calponin 3 was not significantly regulated by mechanical strain or TGF-β1, which was in contrast to the synergistic up-regulation of calponin 1 gene expression by cyclic strain and TGF-β1. Further experiments with cycloheximide treatment suggested that the up-regulation of calponin 3 by cyclic strain was at post-transcriptional level. The results in this study suggest that both mechanical stimulation and TGF-β1 signaling play unique and important roles in the regulation of MSCs at both transcriptional and post-transcriptional levels, and that a precise combination of microenvironmental cues may promote MSC differentiation

Methylation screening of the TGFBI promoter in human lung and prostate cancer by methylation-specific PCR

<p>Abstract</p> <p>Background</p> <p>Hypermethylation of the <it>TGFBI </it>promoter has been shown to correlate with decreased expression of this gene in human tumor cell lines. In this study, we optimized a methylation-specific polymerase chain reaction (MSP) method and investigated the methylation status of the <it>TGFBI </it>promoter in human lung and prostate cancer specimens.</p> <p>Methods</p> <p>Methylation-specific primers were designed based on the methylation profiles of the <it>TGFBI </it>promoter in human tumor cell lines, and MSP conditions were optimized for accurate and efficient amplification. Genomic DNA was isolated from lung tumors and prostatectomy tissues of prostate cancer patients, bisulfite-converted, and analyzed by MSP.</p> <p>Results</p> <p>Among 50 lung cancer samples, 44.0% (22/50) harbored methylated CpG sites in the <it>TGFBI </it>promoter. An analysis correlating gene methylation status with clinicopathological cancer features revealed that dense methylation of the <it>TGFBI </it>promoter was associated with a metastatic phenotype, with 42.9% (6/14) of metastatic lung cancer samples demonstrating dense methylation vs. only 5.6% (2/36) of primary lung cancer samples (<it>p </it>< 0.05). Similar to these lung cancer results, 82.0% (41/50) of prostate cancer samples harbored methylated CpG sites in the <it>TGFBI </it>promoter, and dense methylation of the promoter was present in 38.9% (7/18) of prostate cancer samples with the feature of locoregional invasiveness vs. only 19.4% (6/31) of prostate cancer samples without locoregional invasiveness (<it>p </it>< 0.05). Furthermore, promoter hypermethylation correlated with highly reduced expression of the <it>TGFBI </it>gene in human lung and prostate tumor cell lines.</p> <p>Conclusion</p> <p>We successfully optimized a MSP method for the precise and efficient screening of <it>TGFBI </it>promoter methylation status. Dense methylation of the <it>TGFBI </it>promoter correlated with the extent of <it>TGFBI </it>gene silencing in tumor cell lines and was related to invasiveness of prostate tumors and metastatic status of lung cancer tumors. Thus, <it>TGFBI </it>promoter methylation can be used as a potential prognostic marker for invasiveness and metastasis in prostate and lung cancer patients, respectively.</p

Human U87 Astrocytoma Cell Invasion Induced by Interaction of βig-h3 with Integrin α5β1 Involves Calpain-2

It is known that βig-h3 is involved in the invasive process of many types of tumors, but its mechanism in glioma cells has not been fully clarified. Using immunofluorescent double-staining and confocal imaging analysis, and co-immunoprecipitation assays, we found that βig-h3 co-localized with integrin α5β1 in U87 cells. We sought to elucidate the function of this interaction by performing cell invasion assays and gelatin zymography experiments. We found that siRNA knockdowns of βig-h3 and calpain-2 impaired cell invasion and MMP secretion. Moreover, βig-h3, integrins and calpain-2 are known to be regulated by Ca2+, and they are also involved in tumor cell invasion. Therefore, we further investigated if calpain-2 was relevant to βig-h3-integrin α5β1 interaction to affect U87 cell invasion. Our data showed that βig-h3 co-localized with integrin α5β1 to enhance the invasion of U87 cells, and that calpain-2, is involved in this process, acting as a downstream molecule

Effects of combined treatment with rapamycin and cotylenin A, a novel differentiation-inducing agent, on human breast carcinoma MCF-7 cells and xenografts

INTRODUCTION: Rapamycin, an inhibitor of the serine/threonine kinase target of rapamycin, induces G(1 )arrest and/or apoptosis. Although rapamycin and its analogues are attractive candidates for cancer therapy, their sensitivities with respect to growth inhibition differ markedly among various cancer cells. Using human breast carcinoma cell line MCF-7 as an experimental model system, we examined the growth-inhibitory effects of combinations of various agents and rapamycin to find the agent that most potently enhances the growth-inhibitory effect of rapamycin. METHOD: We evaluated the growth-inhibitory effect of rapamycin plus various agents, including cotylenin A (a novel inducer of differentiation of myeloid leukaemia cells) to MCF-7 cells, using either MTT assay or trypan blue dye exclusion test. The cell cycle was analyzed using propidium iodide-stained nuclei. Expressions of several genes in MCF-7 cells with rapamycin plus cotylenin A were studied using cDNA microarray analysis and RT-PCR. The in vitro results of MCF-7 cells treated with rapamycin plus cotylenin A were further confirmed in vivo in a mouse xenograft model. RESULTS: We found that the sensitivity of rapamycin to MCF-7 cells was markedly affected by cotylenin A. This treatment induced growth arrest of the cells at the G(1 )phase, rather than apoptosis, and induced senescence-associated β-galactosidase activity. We examined the gene expression profiles associated with exposure to rapamycin and cotylenin A using cDNA microarrays. We found that expressions of cyclin G(2), transforming growth factor-β-induced 68 kDa protein, BCL2-interacting killer, and growth factor receptor-bound 7 were markedly induced in MCF-7 cells treated with rapamycin plus cotylenin A. Furthermore, combined treatment with rapamycin and cotylenin A significantly inhibited the growth of MCF-7 cells as xenografts, without apparent adverse effects. CONCLUSION: Rapamycin and cotylenin A cooperatively induced growth arrest in breast carcinoma MCF-7 cells in vitro, and treatment with rapamycin and cotylenin A combined more strongly inhibited the growth of MCF-7 cells as xenografts in vivo than treatment with rapamycin or cotylenin A alone, suggesting that this combination may have therapeutic value in treating breast cancer. We also identified several genes that were markedly modulated in MCF-7 cells treated with rapamycin plus cotylenin A

Established and Newly Proposed Mechanisms of Chronic Cyclosporine Nephropathy

Cyclosporine (CsA) has improved patient and graft survival rates following solid-organ transplantation and has shown significant clinical benefits in the management of autoimmune diseases. However, the clinical use of CsA is often limited by acute or chronic nephropathy, which remains a major problem. Acute nephropathy depends on the dosage of CsA and appears to be caused by a reduction in renal blood flow related to afferent arteriolar vasoconstriction. However, the mechanisms underlying chronic CsA nephropathy are not completely understood. Activation of the intrarenal renin-angiotensin system (RAS), increased release of endothelin-1, dysregulation of nitric oxide (NO) and NO synthase, up-regulation of transforming growth factor-beta1 (TGF-β1), inappropriate apoptosis, stimulation of inflammatory mediators, enhanced innate immunity, endoplasmic reticulum stress, and autophagy have all been implicated in the pathogenesis of chronic CsA nephropathy. Reducing the CsA dosage or using other renoprotective drugs (angiotensin II receptor antagonist, mycophenolate mofetil, and statins, etc.) may ameliorate chronic CsA-induced renal injury. This review discusses old and new concepts in CsA nephropathy and preventive strategies for this clinical dilemma

ALCAM Regulates Motility, Invasiveness, and Adherens Junction Formation in Uveal Melanoma Cells

ALCAM, a member of the immunoglobulin superfamily, has been implicated in numerous developmental events and has been repeatedly identified as a marker for cancer metastasis. Previous studies addressing ALCAM’s role in cancer have, however, yielded conflicting results. Depending on the tumor cell type, ALCAM expression has been reported to be both positively and negatively correlated with cancer progression and metastasis in the literature. To better understand how ALCAM might regulate cancer cell behavior, we utilized a panel of defined uveal melanoma cell lines with high or low ALCAM levels, and directly tested the effects of manipulating these levels on cell motility, invasiveness, and adhesion using multiple assays. ALCAM expression was stably silenced by shRNA knockdown in a high-ALCAM cell line (MUM-2B); the resulting cells displayed reduced motility in gap-closure assays and a reduction in invasiveness as measured by a transwell migration assay. Immunostaining revealed that the silenced cells were defective in the formation of adherens junctions, at which ALCAM colocalizes with N-cadherin and ß-catenin in native cells. Additionally, we stably overexpressed ALCAM in a low-ALCAM cell line (MUM-2C); intriguingly, these cells did not exhibit any increase in motility or invasiveness, indicating that ALCAM is necessary but not sufficient to promote metastasis-associated cell behaviors. In these ALCAM-overexpressing cells, however, recruitment of ß-catenin and N-cadherin to adherens junctions was enhanced. These data confirm a previously suggested role for ALCAM in the regulation of adherens junctions, and also suggest a mechanism by which ALCAM might differentially enhance or decrease invasiveness, depending on the type of cadherin adhesion complexes present in tissues surrounding the primary tumor, and on the cadherin status of the tumor cells themselves