In response to DNA damage, ataxia-telangiectasia mutant and ataxia-telangiectasia and Rad-3 activate p53, resulting in either cell cycle arrest or apoptosis. We report here that DNA damage stimuli, including etoposide (ETOP), adriamycin (ADR), ionizing irradiation (IR), and ultraviolet irradiation (UV) activate ERK1/2 (ERK) mitogen-activated protein kinase in primary (MEF and IMR90), immortalized (NIH3T3) and transformed (MCF-7) cells. ERK activation in response to ETOP was abolished in ATM؊/؊ fibroblasts (GM05823) and was independent of p53. The MEK1 inhibitor PD98059 prevented ERK activation but not p53 stabilization. Maximal ERK activation in response to DNA damage was not attenuated in MEF p53؊/؊ . However, ERK activation contributes to either cell cycle arrest or apoptosis in response to low or high intensity DNA insults, respectively. Inhibition of ERK activation by PD98059 or U0126 attenuated p21 CIP1 induction, resulting in partial release of the G 2 /M cell cycle arrest induced by ETOP. Furthermore, PD98059 or U0126 also strongly attenuated apoptosis induced by high dose ETOP, ADR, or UV. Conversely, enforced activation of ERK by overexpression of MEK-1/Q56P sensitized cells to DNA damage-induced apoptosis. Taken together, these results indicate that DNA damage activates parallel ERK and p53 pathways in an ATM-dependent manner. These pathways might function cooperatively in cell cycle arrest and apoptosis.

OBJECTIVE—Accumulation of glomerular matrix proteins is central to the pathogenesis of diabetic nephropathy, with resident mesangial cells (MCs) known to upregulate matrix protein synthesis in response to high glucose. Because activation of the GTPase RhoA has been implicated in matrix upregulation, we studied its role in induction of the matrix protein fibronectin in diabetic MCs and in vivo in diabetic nephropathy. RESEARCH DESIGN AND METHODS—Glucose (30 mmol/l)-induced RhoA/Rho-kinase, AP-1 activation, and fibronectin upregulation were assessed by immunoblotting, luciferase, electrophoretic mobility shift assay, enzyme-linked immunosorbent assay, real-time PCR, Northern blots, and immunofluorescence. Streptozotocin-induced diabetic rats were treated with the ρ-kinase inhibitor fasudil, which was compared with enalapril, and functional and pathologic parameters were assessed. RESULTS—Glucose led to RhoA and downstream Rho-kinase activation. Mannitol was without effect. Activity of the transcription factor AP-1, increased in diabetic MCs and kidneys, is important in the profibrotic effects of glucose, and this was dependent on Rho-kinase signaling. Upregulation of fibronectin by glucose, shown to be mediated by activator protein-1 (AP-1), was prevented by Rho-kinase inhibition. RhoA siRNA and dominant-negative RhoA also markedly attenuated fibronectin upregulation by high glucose. Applicability of these findings were tested in vivo. Fasudil prevented glomerular fibronectin upregulation, glomerular sclerosis, and proteinuria in diabetic rats, with effectiveness similar to enalapril. CONCLUSIONS—High glucose activates RhoA/Rho-kinase in MCs, leading to downstream AP-1 activation and fibronectin induction. Inhibition of this pathway in vivo prevents the pathologic changes of diabetic nephropathy, supporting a potential role for inhibitors of RhoA/Rho in the treatment of diabetic renal disease.

Background Breast cancer (BC) displays striking genetic, epigenetic and phenotypic diversity. N 6 -methyladenosine (m6A) in mRNA has emerged as a crucial epitranscriptomic modification that controls cancer self-renewal and cell fate. However, the key enzymes of m6A expression and function in human breast carcinogenesis remain unclear. Methods The expression of m6A methylases (METTL3, METTL14 and WTAP) and demethylases (FTO and ALKBH5) were analyzed by using ONCOMINE and The Cancer Genome Atlas databases and in 36 pairs of BC and adjacent non-cancerous tissue. The level of m6A in BC patients was detected by ELISA, and the function of m6A was analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, colony formation assay and transwell assay. The database of bc-GenExMiner v4.0, Kaplan-Meier Plotter and cBioPortal were queried for correlation, mutation and prognosis analysis of BC. Results The m6A methylases and demethylases were dysregulated in several major malignant tumors. Specifically, the expression of all m6A methylases was reduced in BC as compared with normal controls, but the demethylase ALKBH5 was induced in ONCOMINE databases and confirmed in clinical patients. METTL14 expression was positively correlated with METTL3 expression, and both showed high expression in normal breast-like and luminal-A and -B BC. Functionally, reducing m6A expression by overexpressing METTL14 and/or knockdown of ALKBH5 could inhibit breast cell viability, colony formation and cell migration. Furthermore, Kaplan-Meier, meta-analysis and univariate Cox assay showed that the expression of m6A members including METTL3, METTL14, WTAP and FTO but not their gene mutation and amplification, was tightly associated with cancer progression and poor survival. Conclusions Changes of m6A modulators reduced m6A may promote tumorigenesis and predict poor prognosis in BC. Electronic supplementary material The online version of this article (10.1186/s12885-019-5538-z) contains supplementary material, which is available to authorized users.

Background: Diabetic nephropathy (DN) is one of the most serious complications of diabetes and the leading cause of end-stage chronic kidney disease. Currently, there are no effective drugs for treating DN. Therefore, novel and effective strategies to ameliorate DN at the early stage should be identified. This study aimed to explore the effectiveness and underlying mechanisms of human umbilical cord mesenchymal stem cells (UC-MSCs) in DN. Methods: We identified the basic biological properties and examined the multilineage differentiation potential of UC-MSCs. Streptozotocin (STZ)-induced DN rats were infused with 2 × 10 6 UC-MSCs via the tail vein at week 6. After 2 weeks, we measured blood glucose level, levels of renal function parameters in the blood and urine, and cytokine levels in the kidney and blood, and analyzed renal pathological changes after UC-MSC treatment. We also determined the colonization of UC-MSCs in the kidney with or without STZ injection. Moreover, in vitro experiments were performed to analyze cytokine levels of renal tubular epithelial cell lines (NRK-52E, HK2) and human renal glomerular endothelial cell line (hrGECs).

Abstract. Glomerular capillary hypertension is an important determinant of glomerulosclerosis in rats with subtotal renal ablation. Dietary supplementation with L-arginine increases renal nitric oxide (NO) production and limits glomerular injury in this model, and early benefits are seen without altered glomerular capillary pressure. In an in vitro model of hemodynamically mediated signaling, the authors have reported that subjecting MC to cyclic stretch/relaxation activates the mitogen-activated protein kinase p42/44 (Erk) cascade and that NO and cyclic GMP abrogate stretch-induced Erk activation by inducing actin cytoskeletal disassembly. The actin cytoskeleton is regulated by the Rho family of GTPases, including RhoA; therefore, the authors examined the role of RhoA in stretch-induced Erk activation and as an NO target. In primary rat MC subjected to cyclic mechanical strain, RhoA activity was maximally increased (2.4-fold) after 1 min of stretch, and Erk activation temporally followed. The Rho-kinase inhibitor Y-27632 attenuated Erk activation in a dose-dependent manner and prevented stretch-induced actin stress fiber formation. The NO donors S-nitroso-N-acetylpenicillamine and cGMP both inhibited stretch-induced RhoA and Erk activation and stress fiber formation. Infection of MC with the RhoA mutant RhoAAla188, which is resistant to NO-dependent phosphorylation, abrogated the effects of NO and cGMP on stretch-induced Erk activation and stress fiber formation. The authors conclude that the early activation of RhoA is essential for stretch-induced actin stress fiber formation and Erk activation in MC, events which are prevented by NO and cGMP through their action on RhoA. Inhibition of RhoA may thus be a new approach to the prevention of hemodynamically mediated glomerular injury.The partially nephrectomized (Nx) rat is a well-characterized model of chronic renal failure (CRF) in which glomerular capillary hypertension is an important hemodynamic determinant of glomerulosclerosis (1). We and others have shown that L-arginine, an orally administered precursor for nitric oxide (NO) production, prevents the development and progression of renal lesions in this model (2,3). Although NO is an intrarenal vasodilator (4), the early beneficial effects seen with L-arginine occur in the absence of reductions in glomerular capillary pressure (Pgc) (5).Increased Pgc and wall tension are transmitted to resident glomerular cells. In other models of glomerulosclerosis associated with glomerular capillary hypertension such as the uninephrectomized rat treated with deoxycortisone trimethyl acetate (DOCA) and the fawn-hooded rat, podocyte injury is an important determinant of progressive glomerulosclerosis (6,7). However, the behavior of mesangial cells (MC) is thought to play a central role in the progression of glomerular disease in the remnant kidney. Although a rise in Pgc precedes MC proliferation and increased extracellular matrix (ECM) production in the remnant glomerulus (1,8), the mechanisms responsible for linking glo...

Accumulation of glomerular matrix is a hallmark of diabetic nephropathy. The serine/threonine kinase Akt mediates glucose-induced upregulation of collagen I in mesangial cells through transactivation of the EGF receptor (EGFR). In addition, in renal tubular cells, glucose-induced secretion of TGF-␤ requires phosphoinositide-3-OH kinase, suggesting a possible role for Akt in the modulation of TGF-␤ expression, but the mechanisms of Akt activation and its involvement in TGF-␤ regulation are unknown. Here, in primary mesangial cells, high glucose induced AktS473 phosphorylation, which correlates with its activation, in a protein kinase C ␤ (PKC-␤)-dependent manner. Glucose led to PKC-␤1 membrane translocation and association with Akt, and PKC-␤1 immunoprecipitated from glucose-treated cells phosphorylated recombinant Akt on S473. PKC is known to mediate glucose-induced TGF-␤1 upregulation through the transcription factor AP-1; here, inhibitors of phosphoinositide-3-OH kinase, PKC-␤ and Akt, and dominant-negative Akt all prevented glucose-induced activation of AP-1 and upregulation of TGF-␤1. Finally, pharmacologic and dominant negative inhibition of EGFR blocked glucose-induced activation of PKC-␤1, phosphorylation of AktS473, activation of AP-1, and upregulation of TGF-␤1. In vivo, the PKC-␤ inhibitor ruboxistaurin prevented Akt activation in the renal cortex of diabetic rats. In conclusion, PKC-␤1 is an Akt S473 kinase in glucose-treated mesangial cells, and TGF-␤1 transcriptional upregulation requires EGFR/PKC-␤1/Akt signaling. New therapeutic approaches for diabetic nephropathy may result from targeting components of this pathway, particularly the initial EGFR transactivation. 20: 554 -566, 200920: 554 -566, . doi: 10.1681 The kidney is an important site of diabetic microvascular complications, and hyperglycemia is central to glomerular matrix accumulation. Although strict glucose control and inhibition of the reninangiotensin system are effective in delaying the development of nephropathy, disease progression often occurs. The development of new treatment approaches is thus an important goal. J Am Soc NephrolWe have shown that collagen I induction by high glucose (HG) requires activation of the serine/threonine kinase Akt, and this depends on transactivation of the EGF receptor (EGFR). 1 Akt activation requires membrane translocation and phosphorylation on two sites, S473 and T308. 2 Phosphoinositide-3-OH kinase (PI3K) is an upstream mediator of Akt activation, generating phosphorylated lipid second messengers that recruit proteins with pleckstrin homology domains such as Akt to the membrane. 3 At the membrane, phosphoinositide-dependent protein kinase 1 (PDK1) phosphorylates Akt at T308. 3 Several S473 kinases, however, have

Acute kidney injury (AKI) is a common syndrome with a high mortality and morbidity rate. Recent developments in stem cell research have shown great promise for the treatment of AKI. The aim of this study was to investigate the therapeutic potential and anti-apoptotic mechanisms of action of bone marrow-derived mesenchymal stem cells (BM-MSCs) in the treatment of AKI induced by cisplatin in vivo and in vitro. In vivo, adult male Sprague-Dawley rats (n=24) were administered BM-MSCs intravenously one day after cisplatin injection. The rats were sacrificed four days after the cisplatin injection and the effects of BM-MSCs on cisplatin-induced AKI, as well as the anti-apoptotic mechanisms involved were investigated. In vitro, NRK-52E cells, a rat renal proximal tubular cell line, were incubated in conditioned medium or complete medium in the presence or absence of cisplatin, followed by cell proliferation and apoptosis assays. The infusion of BM-MSCs preserved renal function, ameliorated renal tubular lesions, reduced apoptosis and accelerated tubular cell regeneration in the rats with cisplatin-induced AKI. The infusion of BM-MSCs also inhibited the activation of two mitogen-activated protein kinases, p38 and ERK, downregulated the expression of Bax and cleaved caspase-3, and upregulated the expression of Bcl-2. BM-MSC-conditioned medium improved NRK-52E cell viability and inhibited apoptosis. In conclusion, our results demonstrate that injecting rats with BM-MSCs protects renal function and structure in cisplatin-induced AKI by inhibiting cell apoptosis in vivo. BM-MSC-conditioned medium protects renal cells from apoptosis induced by cisplatin in vitro. Hence, the infusion of BM-MSCs should be considered as a possible therapeutic strategy for the treatment of AKI.

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