KH-type Splicing Regulatory Protein Expression Research Articles

KSRP improves pancreatic beta cell function and survival

Impaired insulin production and/or secretion by pancreatic beta cells can lead to high blood glucose levels and type 2 diabetes (T2D). Therefore, investigating new proteins involved in beta cell response to stress conditions could be useful in finding new targets for therapeutic approaches. KH-type splicing regulatory protein (KSRP) is a protein usually involved in gene expression due to its role in post-transcriptional regulation. Although there are studies describing the important role of KSRP in tissues closely related to glucose homeostasis, its effect on pancreatic beta cells has not been explored so far. Pancreatic islets from diet-induced obese mice (C57BL/6JUnib) were used to determine KSRP expression and we also performed in vitro experiments exposing INS-1E cells (pancreatic beta cell line) to different stressors (palmitate or cyclopiazonic acid—CPA) to induce cellular dysfunction. Here we show that KSRP expression is reduced in all the beta cell dysfunction models tested. In addition, when manipulated to knock down KSRP, beta cells exhibited increased death and impaired insulin secretion, whereas KSRP overexpression prevented cell death and increased insulin secretion. Taken together, our findings suggest that KSRP could be an important target to protect beta cells from impaired functioning and death.

Blockage of KHSRP-NLRP3 by MCC950 Can Reverse the Effect of Manganese-Induced Neuroinflammation in N2a Cells and Rat Brain.

Manganese neurotoxicity has been reported to cause a neurodegenerative disease known as parkinsonism. Previous reports have shown that the expression of the KH-type splicing regulatory protein (KHSRP), a nucleic acid-binding protein, and NLRP3 is increased upon Mn exposure. However, the relation between these two during Mn toxicity has not been fully deduced. The mouse neuroblastoma (N2a) and SD rats are treated with LPS and MnCl2 to evaluate the expression of KHSRP and NLRP3. Further, the effect of the NLRP3 inhibitor MCC950 is checked on the expression of NLRP3, KHSRP and pro-inflammatory markers (TNFα, IL-18 and IL-1β) as well as the caspase-1 enzyme. Our results demonstrated an increment in NLRP3 and KHSRP expression post-MnCl2 exposure in N2a cells and rat brain, while on the other hand with LPS exposure only NLRP3 expression levels were elevated and KHSRP was found to be unaffected. An increased expression of KHSRP, NLRP3, pro-inflammatory markers and the caspase-1 enzyme was observed to be inhibited with MCC950 treatment in MnCl2-exposed cells and rats. Manganese exposure induces NLRP3 and KHSRP expression to induce neuroinflammation, suggesting a correlation between both which functions in toxicity-related pathways. Furthermore, MCC950 treatment reversed the role of KHSRP from anti-inflammatory to pro-inflammatory.

KH-Type Splicing Regulatory Protein Controls Colorectal Cancer Cell Growth and Modulates the Tumor Microenvironment

KH-type splicing regulatory protein (KHSRP) is a multifunctional nucleic acid binding protein implicated in key aspects of cancer cell biology: inflammation and cell-fate determination. However, the role KHSRP plays in colorectal cancer (CRC) tumorigenesis remains largely unknown. Using a combination of in silico analysis of large data sets, exvivo analysis of protein expression in patients, and mechanistic studies using invitro models of CRC, we investigated the oncogenic role of KHSRP. We demonstrated KHSRP expression in the epithelial and stromal compartments of both primary and metastatic tumors. Elevated expression was found in tumor versus matched normal tissue, and these findings were validated in larger independent cohorts in silico. KHSRP expression was a prognostic indicator of worse overall survival (hazard ratio,3.74; 95% CI,1.43-22.97; P=0.0138). Mechanistic data in CRC cell line models supported a role of KHSRP in driving epithelial cell proliferation in both a primary and metastatic setting, through control of the G1/S transition. In addition, KHSRP promoted a proangiogenic extracellular environment by regulating the secretion of oncogenic proteins involved in diverse cellular processes, such as migration and response to cellular stress. Our study provides novel mechanistic insight into the tumor-promoting effects of KHSRP in CRC.

Oxymatrine ameliorates insulin resistance in rats with type 2 diabetes by regulating the expression of KSRP, PETN, and AKT in the liver.

Insulin resistance plays a key role in the development and progression of type 2 diabetes mellitus (T2DM). Recent studies found that insulin resistance was associated with the dysfunction of KH-type splicing regulatory protein (KSRP) expression and AKT pathway, and that oxymatrine possesses an antidiabetic effect. The aim of the present study was to investigate whether the protection of oxymatrine against T2DM was associated with the modulation of the KSRP expression and AKT pathway. Sprague-Dawley rats were fed a high-fat diet and injected with streptozotocin intraperitoneally to induce T2DM, which led to an increase in blood glucose levels and insulin resistance, and a decrease in insulin sensitivity and glycogen synthesis concomitant with KSRP downregulation, PTEN upregulation, and AKT phosphorylation deficiency. The administration of oxymatrine decreased blood glucose levels and insulin resistance, increased insulin sensitivity, and improved glycogen synthesis in the liver of T2DM rats, through a reversal in the expression of KSRP, PTEN, and AKT. On the basis of these observations, we concluded that oxymatrine can protect T2DM rats from insulin resistance through the regulation of the KSRP, PETN, and AKT expression in the liver.

Long Noncoding RNA AB074169 Inhibits Cell Proliferation via Modulation of KHSRP-Mediated CDKN1a Expression in Papillary Thyroid Carcinoma.

Long noncoding RNAs (lncRNA) are emerging as a novel class of regulators in gene expression associated with tumorigenesis. However, the role of lncRNAs in papillary thyroid carcinoma (PTC) is poorly understood. Here, we conducted global lncRNA profiling and identified lncRNA AB074169 (lncAB) as significantly downregulated in PTC. Decreased expression of lncAB in PTC was caused by CpG hypermethylation within its gene promoter. Functional studies showed that lncAB overexpression led to cell-cycle arrest and tumor growth inhibition in vitro and in vivo, whereas lncAB knockdown promoted cell proliferation. Mechanistic analyses revealed that lncAB bound KH-type splicing regulatory protein (KHSRP) and also decreased expression of KHSRP, thus increasing CDKN1a (p21) expression and decreasing CDK2 expression to repress cell proliferation. Taken together, these findings demonstrate that lncAB functions as a tumor suppressor during PTC tumorigenesis.Significance: These findings identify a tumor-suppressive long noncoding RNA in papillary thyroid carcinoma.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/15/4163/F1.large.jpg Cancer Res; 78(15); 4163-74. ©2018 AACR.

KH-type splicing regulatory protein is regulated by nuclear factor-κB signaling to mediate innate immunity in Caco-2 cells infected by Salmonella enteritidis.

Salmonella enteritidis infection occurs in enterogenous diseases, such as gastroenteritis and parenteral focal infection, which often involve inflammation of intestinal epithelial cells. The nuclear factor kappa B (NF-κB) pathway participates in the innate immune response to many gram-negative pathogenic bacteria and initiates inflammation in epithelial cells. KH-type splicing regulatory protein (KSRP) is a multi-domain RNA-binding protein that recruits the exosome-containing mRNA degradation complex to mRNAs coding for inflammatory response factors. However, it remains unclear whether KSRP is regulated by NF-κB signaling pathway in response to S. enteritidis infection and affects the development of inflammation. Accordingly, in this study, we investigated the role of KSRP in mediating the response to S. enteritidis in Caco-2 cells. The data revealed that S. enteritidis infection decreased KSRP expression, which was suppressed by blocking the NF-κB pathway. Additionally, S. enteritidis infection significantly increased the expression of inducible nitric oxide synthase and cyclooxygenase-2. Overexpression of KSRP reduced the expression levels of inflammatory factors in Caco-2 cells. KSRP was regulated by the NF-κB signaling pathway and participated in mediating the innate immune response to S. enteritidis infection in Caco-2 cells, and KSRP acted as a negative regulator of inflammatory gene expression.

Effects of the RNA-binding protein, KSRP, on innate immune response against Helicobacter pylori infection in mice

Helicobacter pylori (H. pylori) contributes to various gastric diseases such as chronic gastritis, gastric ulcer, and gastric carcinoma. Host innate immune response against the pathogen plays a significant role in elimination of pathogen infection. Importantly, pathogen elimination is closely related to numerous inflammatory-related genes that participate in complex biological response of cells to harmful stimuli. Here we studied effects of the KH-type splicing regulatory protein (KSRP), a RNA-binding protein, on innate immune response against H. pylori infection. We found that H. pylori infection downregulated KSRP expression directly, and that KSRP overexpression repressed upregulation of CXCL-2 expression induced by H. pylori and facilitated H. pylori proliferation in vitro. Similarly, KSRP overexpression in H. pylori mice also facilitated H. pylori proliferation and colonization, and induced more severe gastric mucosal damage. Intriguingly, CXCL-2 and HMOX-1 were upregulated in H. pylori infected mice after KSRP overexpression. This difference in expression of these genes implicated that KSRP was closely associated with and directly participated in the innate immune response against H. pylori. These results were beneficial for understanding the in vivo function of KSRP on innate immune response against pathogen infection.

KH-type splicing regulatory protein is involved in esophageal squamous cell carcinoma progression.

KH-type splicing regulatory protein (KHSRP) is a multifunctional RNA-binding protein, which is involved in several post-transcriptional aspects of RNA metabolism, including microRNA (miRNA) biogenesis. It affects distinct cell functions in different tissues and can have an impact on various pathological conditions. In the present study, we investigated the oncogenic functions of KHSRP and their underlying mechanisms in the pathogenesis of esophageal squamous cell carcinoma (ESCC). KHSRP expression levels were elevated in ESCC tumors when compared with those in non-tumorous tissues by immunohistochemistry, and cytoplasmic KHSRP overexpression was found to be an independent prognosticator for worse overall survival in a cohort of 104 patients with ESCC. KHSRP knockdown inhibited growth, migration, and invasion of ESCC cells. KHSRP knockdown also inhibited the maturation of cancer-associated miRNAs, such as miR-21, miR-130b, and miR-301, and induced the expression of their target mRNAs, such as BMP6, PDCD4, and TIMP3, resulting in the inhibition of epithelial-to-mesenchymal transition. Our findings uncover a novel oncogenic function of KHSRP in esophageal tumorigenesis and implicate its use as a marker for prognostic evaluation and as a putative therapeutic target in ESCC.

KSRP suppresses cell invasion and metastasis through miR-23a-mediated EGR3 mRNA degradation in non-small cell lung cancer

KH-type splicing regulatory protein (KSRP) is a single-strand RNA binding protein which regulates mRNA stability either by binding to AU-rich elements (AREs) of mRNA 3′UTR or by facilitating miRNA biogenesis to target mRNA. Unlike its well-characterized function at the molecular level in maintaining RNA homeostasis, the role of KSRP in cancer progression remains largely unknown. Here we investigate the role of KSRP in non-small cell lung cancer (NSCLC). We first examined KSRP expression by immunohistochemistry in a cohort containing 196 NSCLC patients and observed a strong positive correlation between KSRP expression and survival of NSCLC patients. Multivariate analysis further identified KSRP as an independent prognostic factor. Manipulating KSRP expression significantly affected in vitro cell mobility and in vivo metastatic ability of NSCLC cells. Microarray analysis identified an ARE-containing gene, EGR3, as a downstream effector of KSRP in NSCLC. Interestingly, we found that KSRP decreased EGR3 mRNA stability in an ARE-independent manner. By screening KSRP-regulated miRNAs in NSCLC cells, we further found that miR-23a directly binds to EGR3 3′UTR, reducing EGR3 expression and thereby inhibiting NSCLC cell mobility. Our findings implicate a targetable KSRP/miR-23a/EGR3 signaling axis in advanced tumor phenotypes.

KH-type splicing regulatory protein mediate inflammatory response in gastric epithelial cells induced by lipopolysaccharide.

To study differential expressions of KH-type splicing regulatory protein (KSRP) and inflammatory factors and to explore the relationship between them in Lipopolysaccharide (LPS)-induced gastric epithelial cells (GES-1), cells were exposed to LPS for 24 h in the presence or absence of SC-514. Western blot and real-time PCR (RT-PCR) were used to analysis the contents of KSRP, inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2). The results showed that LPS decreased the expression of KSRP protein in GES-1 cells, but not KSRP mRNA, while increasing the levels of iNOS and COX-2 proteins and mRNAs in GES-1cells. High expression of KSRP induced low expressions and stabilities of iNOS and COX-2 in GES-1 cells, indicated that KSRP protein presented negative correlation with iNOS and COX-2 with LPS stimulation. In conclusion, the regulation of expression of KSRP was mainly achieved through post-translational modification. KSRP protein participated in regulating the expression of iNOS and COX-2 in their transcription and translation levels. In response to LPS or gram negative pathogenic microorganism, KSRP could regulate Toll-like receptor (TLR)/ Nuclear factor-kappa B (NF-κB) signal pathway in GES-1 cells.

Upregulation of KSRP by miR-27b provides IFN-γ-induced post-transcriptional regulation of CX3CL1 in liver epithelial cells.

Aberrant cellular responses to pro-inflammatory cytokines, such as IFN-γ, are pathogenic features in many chronic inflammatory diseases. A variety of feedback regulatory pathways have evolved to prevent an inappropriate cellular reaction to these pro-inflammatory cytokines. CX3CL1 is a unique chemokine and plays an important role in chronic liver diseases. We report here that IFN-γ stimulation induces a transient CX3CL1 production in liver epithelial cells (i.e., hepatocytes and biliary epithelial cells). This transient CX3CL1 production is accompanied with a destabilization of CX3CL1 mRNA associated with the induction of the KH-type splicing regulatory protein (KSRP). IFN-γ treatment of liver epithelial cells decreases expression level of miR-27b, a miRNA that targets the 3′ untranslated region of KSRP mRNA resulting in translational suppression. Induction of KSRP following IFN-γ stimulation depends on the downregulation of miR-27b. Functional manipulation of KSRP or miR-27b caused reciprocal alterations in CX3CL1 mRNA stability in liver epithelial cells. Moreover, transfection of miR-27b precursor influences CX3CL1-associated chemotaxis effects of biliary epithelial cells to Jurkat T cells. These findings suggest that miR-27b-mediated post-transcriptional suppression controls the expression of KSRP in liver epithelial cells, and upregulation of KSRP destabilizes CX3CL1 mRNA, providing fine-tuning of cellular inflammatory reactions in response to IFN-γ stimulation.

Bone Morphogenetic Protein/SMAD Signaling Orients Cell Fate Decision by Impairing KSRP-Dependent MicroRNA Maturation

MicroRNAs (miRNAs) are essential regulators of development, physiology, and evolution, and their biogenesis is strictly controlled at multiple levels. Regulatory proteins, such as KSRP, modulate rates and timing of enzymatic reactions responsible for maturation of select miRNAs from their primary transcripts in response to specific stimuli. Here, we show that KSRP silencing in mesenchymal C2C12 cells produces a change in the transcriptome largely overlapping that induced by bone morphogenetic protein 2 (BMP2) signaling activation. This induces osteoblastic differentiation while preventing myogenic differentiation. KSRP silencing- and BMP2-dependent myogenic miRNA (myomiR) maturation blockade is required for osteoblastic differentiation of C2C12 cells. Our results demonstrate that phosphorylated R-SMAD proteins, the transducers of BMP2 signal, associate with phosphorylated KSRP and block its interaction with primary myomiRs. This abrogates KSRP-dependent myomiR maturation, with SMAD4, SMAD5, and SMAD9 silencing being able to rescue KSRP function. Thus, SMAD-induced blockade of KSRP-dependent myomiR maturation is critical for orienting C2C12 cell differentiation toward osteoblastic lineage.

MiR-27b Targets KSRP to Coordinate TLR4-Mediated Epithelial Defense against Cryptosporidium parvum Infection

Cryptosporidium is a protozoan parasite that infects the gastrointestinal epithelium and causes a diarrheal disease. Toll-like receptor (TLR)- and NF-κB-mediated immune responses from epithelial cells, such as production of antimicrobial peptides and generation of reactive nitrogen species, are important components of the host's defense against cryptosporidial infection. Here we report data demonstrating a role for miR-27b in the regulation of TLR4/NF-κB-mediated epithelial anti-Cryptosporidium parvum responses. We found that C. parvum infection induced nitric oxide (NO) production in host epithelial cells in a TLR4/NF-κB-dependent manner, with the involvement of the stabilization of inducible NO synthase (iNOS) mRNA. C. parvum infection of epithelial cells activated NF-κB signaling to increase transcription of the miR-27b gene. Meanwhile, downregulation of KH-type splicing regulatory protein (KSRP) was detected in epithelial cells following C. parvum infection. Importantly, miR-27b targeted the 3′-untranslated region of KSRP, resulting in translational suppression. C. parvum infection decreased KSRP expression through upregulating miR-27b. Functional manipulation of KSRP or miR-27b caused reciprocal alterations in iNOS mRNA stability in infected cells. Forced expression of KSRP and inhibition of miR-27b resulted in an increased burden of C. parvum infection. Downregulation of KSRP through upregulating miR-27b was also detected in epithelial cells following LPS stimulation. These data suggest that miR-27b targets KSRP and modulates iNOS mRNA stability following C. parvum infection, a process that may be relevant to the regulation of epithelial anti-microbial defense in general.

Involvement of KSRP in the post-transcriptional regulation of human iNOS expression-complex interplay of KSRP with TTP and HuR

We purified the KH-type splicing regulatory protein (KSRP) as a protein interacting with the 3′-untranslated region (3′-UTR) of the human inducible nitric oxide (iNOS) mRNA. Immunodepletion of KSRP enhanced iNOS 3′-UTR RNA stability in in vitro-degradation assays. In DLD-1 cells overexpressing KSRP cytokine-induced iNOS expression was markedly reduced. In accordance, downregulation of KSRP expression increases iNOS expression by stabilizing iNOS mRNA. Co-immunoprecipitations showed interaction of KSRP with the exosome and tristetraprolin (TTP). To analyze the role of KSRP binding to the 3′-UTR we studied iNOS expression in DLD-1 cells overexpressing a non-binding mutant of KSRP. In these cells, iNOS expression was increased. Mapping of the binding site revealed KSRP interacting with the most 3′-located AU-rich element (ARE) of the human iNOS mRNA. This sequence is also the target for HuR, an iNOS mRNA stabilizing protein. We were able to demonstrate that KSRP and HuR compete for this binding site, and that intracellular binding to the iNOS mRNA was reduced for KSRP and enhanced for HuR after cytokine treatment. Finally, a complex interplay of KSRP with TTP and HuR seems to be essential for iNOS mRNA stabilization after cytokine stimulation.