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Competing Interests: The author reports no conflicts of interest in this communication.

Esophageal squamous cell carcinoma is a severe malignancy for its high mortality and poor prognosis. Mainstay chemotherapies cause serious side effects for their ways of inducing cell death. Oridonin is the main bioactive constituent from natural plants that has anticancer ability and weak side effects. The proteomics method is efficient to understand the anticancer mechanism. However, proteins identified by proteomics aimed at understanding oridonin's anticancer mechanism is seldom overlapped by different groups. This study used proteomics based on two-dimensional electrophoresis sodium dodecyl sulfate-polyacrylamide gel electrophoresis (2-DE SDS-PAGE) integrated with mass spectrometry and Gene Set Enrichment Analysis (GSEA) to understand the anticancer mechanism of oridonin on esophageal squamous cell carcinoma (ESCC). The results showed that oridonin induced ESCC cell death via apoptosis by decreasing the protein expression of LASP1 and PDLIM1.

Tubulointerstitial fibrosis is a common pathway of chronic kidney disease (CKD) and is closely related to the progression of CKD. LMCD1, acting as an intermediary, has been reported to play a role in cardiac fibrosis. However, its role in renal fibrosis is yet to be deciphered. Based on the GEO database, we found the expression of LMCD1 is increased in kidney tissues of CKD patients and in human proximal tubular epithelial (HK-2) cells treated with transforming growth factor-β1 (TGF-β1), suggesting that LMCD1 may be involved in tubulointerstitial fibrosis. Herein, we investigated the role of LMCD1 in mice with unilateral ureteral obstruction (UUO) and in TGF-β1-stimulated HK-2 cells. In the UUO model, the expression of LMCD1 was upregulated. UUO-induced renal histopathological changes were mitigated by knockdown of LMCD1. LMCD1 silence alleviated renal interstitial fibrosis in UUO mice by decreasing the expression of TGF-β1, fibronectin, collagen I, and collagen III. LMCD1 deficiency suppressed cell apoptosis in kidney to prevent UUO-triggered renal injury. Furthermore, LMCD1 deficiency blocked the activation of ERK signaling in UUO mice. In vitro, LMCD1 was upregulated in HK-2 cells after TGF-β1 stimulation. LMCD1 silence abrogated TGF-β1-mediated upregulation of fibrotic genes. Treatment of HK-2 cells with ERK-specific inhibitor SCH772984 and agonist TPA validated LMCD1 exerted its function via activating ERK signaling. Together, our findings suggest that inhibition of LMCD1 protects against renal interstitial fibrosis by impeding ERK activation., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Planar cell polarity (PCP) signaling regulates several polarization events during development of ommatidia in the Drosophila eye, including directing chirality by polarizing a cell fate choice and determining the direction and extent of ommatidial rotation. The pksple isoform of the PCP protein Prickle is known to participate in the R3/R4 cell fate decision, but the control of other polarization events and the potential contributions of the three Pk isoforms have not been clarified. Here, by characterizing expression and subcellular localization of individual isoforms together with re-analyzing isoform specific phenotypes, we show that the R3/R4 fate decision, its coordination with rotation direction, and completion of rotation to a final ±90° rotation angle are separable polarization decisions with distinct Pk isoform requirements and contributions. Both pksple and pkpk can enforce robust R3/R4 fate decisions, but only pksple can correctly orient them along the dorsal-ventral axis. In contrast, pksple and pkpk can fully and interchangeably sustain coordination of rotation direction and rotation to completion. We propose that expression dynamics and competitive interactions determine isoform participation in these processes. We propose that the selective requirement for pksple to orient the R3/R4 decision and their interchangeability for coordination and completion of rotation reflects their previously described differential interaction with the Fat/Dachsous system which is known to be required for orientation of R3/R4 decisions but not for coordination or completion of rotation., Competing Interests: The authors have declared that no competing interests exist.

Objective: Flavopereirine has been identified to be a potential anti-cancer agent in several types of human cancer. This study aimed to investigate the anti-cancer activity of flavopereirine in oral cancer., Methods: The effect of flavopereirine on cell viability of human oral cancer cell lines (BcaCD885 and Tca8113) was evaluated by MTT assay and colony formation assay. Cell apoptosis and cell cycle distribution were detected by flow cytometry. Cell invasion and migration were evaluated by Transwell assay. The expression of LASP1, JAK2, p-JAK2, STST3, p-STST3, STST5 and p-STST5 was evaluated by qRT-PCR and Western blot. In addition, the xenograft mouse model was constructed to determine the anti-cancer role of flavopereirine in vivo., Results: Flavopereirine significantly inhibited cell proliferation, invasion, migration and EMT process of BcaCD885 and Tca8113 cells, while promoted cell apoptosis in vitro. Flavopereirine markedly decreased the expression levels of p-JAK2, p-STST3 and p-STST5, while increased the expression levels of LASP1. In addition, downregulation of LASP1 significantly increased the expression levels of p-JAK2, p-STAT3 and p-STAT5 compared with si-NC in BcaCD885 cells. Moreover, flavopereirine was found to decrease tumor weight and volume of xenograft tumors in vivo., Conclusion: Flavopereirine inhibited the progression of oral cancer through inactivating the JAK/STAT signaling pathway by upregulating LASP1, suggesting that flavopereirine might be a potential anti-cancer agent for oral cancer., Competing Interests: The authors declare that they have no competing interests., (© 2021 Xu et al.)

Accumulating evidence has suggested that thyroid hormone receptor interacting protein 6 (TRIP6) is a novel tumor-related regulator that is aberrantly expressed in multiple tumors and contributes to tumor progression and metastasis. Yet, little is known about the role of TRIP6 in cervical cancer. In the current study, we aimed to explore the expression, biological function, and regulatory mechanism of TRIP6 in cervical cancer. Here we showed that TRIP6 expression was markedly upregulated in cervical cancer tissues and cell lines. The knockdown of TRIP6 suppressed the proliferation, colony formation, and invasive potential of cervical cancer cells, whereas TRIP6 overexpression exhibited the opposite effect. Moreover, TRIP6 contributes to the activation of Yes-associated protein (YAP) by downregulating the level of YAP phosphorylation. Notably, TRIP6-mediated tumor promotion effect was partially reversed by YAP inhibition. In addition, TRIP6 knockdown retarded the in vivo tumor growth of cervical cancer of mouse xenograft models associated with downregulation of YAP activation in tumor tissues. Taken together, these results reveal a potential tumor promotion role of TRIP6 that facilitates the proliferation and invasion of cervical cancer through activation of YAP. Our study underlines the importance of the TRIP6/YAP axis in cervical cancer and suggests TRIP6 as a potential anticancer candidate for cervical cancer., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: Acute myeloid leukaemia (AML) is a malignant haematological tumour with high heterogeneity and mortality. A reliable prognostic assessment is critical for treatment strategies. However, the current prognostic evaluation system of AML is insufficient., Methods: Genome-wide univariate Cox regression analysis was performed on three independent AML datasets to screen for the prognostic-related genes. Kaplan-Meier survival analysis was employed to verify the efficacy of FHL1 in evaluating overall survival in 1298 de novo AML patients, 648 non-acute promyelocytic leukaemia AML patients and 407 cytogenetically normal AML patients; the data for some of these patients were also used for EFS and RFS validation. Multivariate Cox regression was performed to validate FHL1 as an independent prognostic indicator. WGCNA, GSEA, and gene correlation analysis were applied to explore the mechanism of FHL1 in AML. The synergistic cytocidal effect of FHL1 knockdown was verified in in vitro experiments., Findings: Comprehensive genome-wide analyses and large-sample validation showed that FHL1 is a powerful prognostic candidate for overall survival, event-free survival, and relapse-free survival in AML and is independent of prognosis-related clinical factors and genetic abnormalities. The molecular mechanism may occur through regulation of FHL1 in leukaemia stem cells, tumour-associated signalling pathways, and transmembrane transport of chemotherapeutic drugs. FHL1-targeted intervention enhances the sensitivity of AML cells to cytarabine., Interpretation: FHL1 may serve as an evaluation factor for clinical strategy selection, and its targeted intervention may be beneficial for chemotherapy in AML patients., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

LIM and SH3 protein 2 (LASP2) belongs to nebulin family. It has been proven that LASP2 is involved in several cancers; however, its role in cervical cancer is unclear. Herein, we showed that LASP2 was highly expressed in cervical cancer tissues and cell lines. To knockdown LASP2 in cervical cancer cells, small interfering RNAs (siRNAs) targeting LASP2 (si-LASP2) were used. We found that cell proliferation, migration/invasion were markedly reduced after si-LASP2 transfection. A significant increase in E-cadherin expression, and decrease in N-cadherin and vimentin expressions were observed in si-LASP2 transfected cervical cancer cells. Knockdown of LASP2 caused significant inhibitory effect on the PI3K/Akt pathway. Treatment with the activator of the PI3K/Akt pathway, 740Y-P, abolished the effects of si-LASP2 transfection on cervical cancer cells. These findings suggested that LASP2 may be an oncogene through regulating the PI3K/Akt pathway in cervical cancer., (© 2019 Wiley Periodicals, Inc.)

Aberrant long noncoding RNAs (lncRNA) have been proved to be associated with the many types of malignant tumors (including hepatocellular carcinoma [HCC]). In this study, a lncRNAs and mRNAs microarray analysis was performed in three pairs of HCC patitents' tumor. We found lncRNA LIM and SH3 protein 1 antisense (LASP1-AS) and its sense-cognate gene LIM and SH3 protein 1 (LASP1) were upregulated in HCC and both are correlated with poorer prognosis and lower survival of HCC patients. Meanwhile, the expression of LASP1-AS correlated positively with LASP1 expression in HCC tissues. LASP1-AS promoted the proliferation, migration, and invasion abilities of HCC in vitro and vivo by enhancing LASP1 expression. Our study explored lncRNA LASP1-AS as an oncogene in HCC and promoted proliferation and metastasis capabilities of HCC via increasing the expression of its sense-cognate gene LASP1. LncRNA LASP1-AS might be a potential valuable prognostic biomarker and potential therapeutic target of HCC., (© 2019 Wiley Periodicals, Inc.)

Purpose: Oxygen and glucose deprivation is a common feature of the solid tumor. Regulatory network underlying the adaptation of cancer cells to the harsh microenvironment remains unclear. We determined the mechanistic role of LIM and senescent cell antigen-like-containing domain protein 1 (LIMS1) in cancer cell survival under oxygen-glucose deprivation conditions., Experimental Design: The expression level of LIMS1 was determined by IHC staining and analyzing the mRNA expression profiles from The Cancer Genome Atlas of three human solid tumors. Roles of LIMS1 in cancer cell metabolism and growth were determined by molecular and cell biology methods. A jetPEI nanocarrier was used as the vehicle for anti-LIMS1 siRNAs in mouse models of cancer therapeutics., Results: LIMS1 expression was drastically elevated in pancreatic ductal adenocarcinoma (PDAC). High LIMS1 level was associated with advanced TNM stage and poor prognosis of patients with tumor. Increased LIMS1 expression was pivotal for tumor cells to survive in the oxygen-glucose deprivation conditions. Mechanistically, LIMS1 enhanced GLUT1 expression and membrane translocation, which facilitated tumor cell adaptation to the glucose deprivation stress. Furthermore, LIMS1 promoted HIF1A protein translation by activating AKT/mTOR signaling, while hypoxia-inducible factor 1 (HIF1) transactivated LIMS1 transcription, thus forming a positive feedback loop in PDAC cell adaptation to oxygen deprivation stress. Inhibition of LIMS1 with jetPEI nanocarrier-delivered anti-LIMS1 siRNAs significantly increased cell death and suppressed tumor growth., Conclusions: LIMS1 promotes pancreatic cancer cell survival under oxygen-glucose deprivation conditions by activating AKT/mTOR signaling and enhancing HIF1A protein translation. LIMS1 is crucial for tumor adaptation to oxygen-glucose deprivation conditions and is a promising therapeutic target for cancer treatment., (©2019 American Association for Cancer Research.)

Background: LIM and SH3 protein 1 (LASP1) is upregulated in several types of human cancer and implicated in cancer progression. However, the expression and intrinsic function of LASP1 in glioblastoma (GBM) remains unclear., Method: Oncomine and The Cancer Genome Atlas (TCGA) database was analyzed for the expression and clinical significance of LASP1 in GBM. LASP1 mRNA and protein level were measured by qRT-PCR and western blotting. The effect of LASP1 on GBM proliferation was examined by MTT assay and colony formation assay, the effect of LASP1 on sensitivity of Temozolomide was measured by flow cytometry and subcutaneous tumor model. The association between LASP1 and PI3K/AKT signaling was assessed by western blotting., Results: Oncomine GBM dataset analysis indicated LASP1 is significantly upregulated in GBM tissues compared to normal tissues. GBM dataset from The Cancer Genome Atlas (TCGA) revealed that high LASP1 expression is related to poor overall survival. LASP1 mRNA and protein in clinical specimens and tumor cell lines are frequently overexpressed. LASP1 knockdown dramatically suppressed U87 and U251 cell proliferation. Silencing LASP1 potentiated cell chemosensitivity to temozolomide in vitro, LASP1 knockdown inhibited tumor growth and enhanced the therapeutic effect of temozolomide in vivo. TCGA dataset analysis indicated LASP1 was correlated with PI3K/AKT signaling pathway, and LASP1 deletion inhibited this pathway. Combination treatment with PI3K/AKT pathway inhibitor LY294002 dramatically accelerated the suppression effect of temozolomide., Conclusion: LASP1 may function as an oncogene in GBM and regulate cell proliferation and chemosensitivity in a PI3K/AKT-dependent mechanism. Thus, the LASP1/PI3K/AKT axis is a promising target and therapeutic strategy for GBM treatment.

Actin-binding LIM protein 1 (ABLIM1), a member of the LIM-domain protein family, mediates interactions between actin filaments and cytoplasmic targets. However, the role of ABLIM1 in osteoclast and bone metabolism has not been reported. In the present study, we investigated the role of ABLIM1 in the receptor activator of NF-κB ligand (RANKL)- mediated osteoclastogenesis. ABLIM1 expression was induced by RANKL treatment and knockdown of ABLIM1 by retrovirus infection containing Ablim1-specific short hairpin RNA (shAblim1) decreased mature osteoclast formation and bone resorption activity in a RANKL-dose dependent manner. Coincident with the downregulated expression of osteoclast differentiation marker genes, the expression levels of c-Fos and the nuclear factor of activated T-cells cytoplasmic 1 (NFATc1), critical transcription factors of osteoclastogenesis, were also decreased in shAblim1-infected osteoclasts during RANKLmediated osteoclast differentiation. In addition, the motility of preosteoclast was reduced by ABLIM1 knockdown via modulation of the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/Akt/Rac1 signaling pathway, suggesting another regulatory mechanism of ABLIM1 in osteoclast formation. These data demonstrated that ABLIM1 is a positive regulator of RANKLmediated osteoclast formation via the modulation of the differentiation and PI3K/Akt/Rac1-dependent motility. [BMB Reports 2018; 51(7): 356-361].

Background/aim: Overall survival for the high-risk group of neuroblastoma (NB) remains at 40-50%. An integrative genomics study revealed that LIM domain only 1 (LMO1) encoding a transcriptional regulator to be an NB-susceptibility gene with a tumor-promoting activity, that needs to be revealed., Materials and Methods: We conducted chromatin immunoprecipitation and DNA sequencing analyses and cell proliferation assays on two NB cell lines., Results: We identified three genes regulated by LMO1 in the cells, LIM and senescent cell antigen-like domains 1 (LIMS1), Ras suppressor protein 1 (RSU1) and relaxin 2 (RLN2). LIMS1 and RSU1 encode proteins functioning with integrin-linked kinase (ILK), and inhibition of LIMS1, ILK or RLN2 by shRNA reduced cell proliferation of the NB cells, which was also suppressed with an ILK inhibiting compound Cpd 22., Conclusion: The downstream of LMO1-regulatory cascade includes a tumor-promoting LIMS1/ILK pathway, which has a potential to be a novel therapeutic target., (Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Actin binding LIM 1 (abLIM1) is a cytoskeletal actin-binding protein that has been implicated in interactions between actin filaments and cytoplasmic targets. Previous biochemical and cytochemical studies have shown that abLIM1 interacts and co-localizes with F-actin in the retina and muscle. However, whether abLIM1 regulates osteoclast differentiation has not yet been elucidated. In this study, we examined the role of abLIM1 in osteoclast differentiation and function. We found that abLIM1 expression was upregulated during receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclast differentiation, and that a novel transcript of abLIM1 was exclusively expressed in osteoclasts. Overexpression of abLIM1 in the murine monocytic cell line, RAW-D suppressed osteoclast differentiation and decreased expression of several osteoclast-marker genes. By contrast, small interfering RNA-induced knockdown of abLIM1 enhanced the formation of multinucleated osteoclasts and markedly increased the expression of the osteoclast-marker genes. Mechanistically, abLIM1 regulated the localization of tubulin, migration, and fusion in osteoclasts. Thus, these results indicate that abLIM1 negatively controls osteoclast differentiation by regulating cell migration and fusion mediated via actin formation., (© 2018 Wiley Periodicals, Inc.)

Background/aims: The malignant biological behavior of gastric cancer(GC) is not only determined by cancer cells alone, but also closely regulated by the microenvironment. Fibroblasts represent a large proportion of the components in the tumor microenvironment, and they promote the development of disease. Currently, accumulating evidence suggests that exosomes can function as intercellular transport systems to relay their contents, especially microRNAs(miRNAs)., Methods: First, we detected the highly-expressed level of miR-27a in exosomes isolated from gastric cancer cells by qRT-PCR. MiR-27a -over-expressed models in vitro and in vivo were established to investigate the transformation of cancer-associated fibroblasts observed by Western blotting, and the malignant behavior of gastric cancer cells using the methods CCK8 and Transwell. Moreover, the downregulation of CSRP2 in fibroblasts was used to evaluate the promotion of malignancy of gastric cancer using the methods CCK8 and Transwell., Results: In this study, we found a marked high level of miR-27a in exosomes derived from GC cells. miR-27a was found to function an oncogene that not only induced the reprogramming of fibroblasts into cancer-associated fibroblasts(CAFs), but also promoted the proliferation, motility and metastasis of cancer cells in vitro and in vivo. Conversely, CAFs with over-expression of miR-27a could pleiotropically increase the malignant behavior of the GC cells. For the first time, we revealed that CSRP2 is a downstream target of miR-27a. CSRP2 downregulation could increase the proliferation and motility of GC cells., Conclusion: Thus, this report indicates that miR-27a in exosomes derived from GC cells has a crucial impact on the microenvironment and may be used as a potential therapeutic target in the treatment of GC., (© 2018 The Author(s). Published by S. Karger AG, Basel.)

LMO7 (LIM domain only 7) is a transcription regulator for expression of many Emery-Dreifuss muscular dystrophy-relevant genes, and binds to α-actinin and AF6/afadin at adherens junctions for epithelial cell-cell adhesion. In this study, we found that human LMO7 interacted with the spindle assembly checkpoint (SAC) protein MAD1. LMO7 colocalized with actin filaments at the cell membrane but did not colocalize with MAD1 at kinetochores in prometaphase. Our observations reveal that overexpression but not depletion of LMO7 caused a SAC defect, and that the LIM domain of LMO7 was a determinant of its ability to interfere with kinetochore localization of the SAC proteins MAD2 and BUBR1 and cause a SAC defect though the LIM peptide itself did neither bind to MAD1, MAD2 and BUBR1 nor localize to the actin filaments. However, overexpression of LMO7 or the LIM peptide did not interfere with kinetochore localization of MAD1. Additionally, overexpression of the LIM peptide prolonged mitotic timing and interfered with chromosome congression whereas that of LMO7b did not. Taken together, we conclude that LMO7 via its LIM domain acts to control mitosis progression and exerts an effect on the SAC., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Research on neural stem cells (NSCs) has recently focused on microRNAs (miRNAs), a class of small non‑coding RNAs that have crucial roles in regulating NSC proliferation and differentiation. In the present study, a quantitative‑polymerase chain reaction assay revealed that the expression of miRNA (miR)‑138‑5p was significantly decreased during neural differentiation of NSCs in vitro. Overexpression of miR‑138‑5p reduced NSC proliferation and increased NSC differentiation. Furthermore, suppression of miR‑138‑5p via transfection with a miRNA inhibitor enhanced NSC proliferation and attenuated NSC differentiation. Additionally, expression of thyroid hormone receptor interacting protein 6 (TRIP6), a critical regulator of NSCs, was negatively correlated with the miR‑138‑5p level. A luciferase assay demonstrated that miR‑138‑5p regulate TRIP6 by directly binding the 3'‑untranslated region of the mRNA. Additionally, upregulation of TRIP6 rescued the NSC proliferation deficiency induced by miR‑138‑5p and abolished miR‑138‑5p‑promoted NSCs differentiation. By contrast, downregulation of TRIP6 produced the opposite effect on proliferation and differentiation of NSCs transfected with anti‑miR‑138‑5p. Taken together, the data suggest that miR‑138‑5p regulates NSCs proliferation and differentiation, and may be useful in developing novel treatments for neurological disorders via manipulation of miR‑138‑5p in NSCs.

Zinc finger protein 185 (ZNF185) belongs to the ZNF family and is involved in cell proliferation and differentiation. To the best of our knowledge, the association between ZNF185 and male reproduction is unknown. In the present study, the expression and localization of ZNF185 in mouse testis, as well as its role in testosterone secretion, cell cycle progression and apoptosis of mouse Leydig cells were investigated. The results of the immunofluorescence analysis indicated that ZNF185 was highly expressed in Leydig cells of the mouse testis, and primarily localized in the cytoplasm. The results of quantitative polymerase chain reaction and western blot analyses further validated that ZNF185 expression was significantly higher in Leydig cells and sperm compared with that in Sertoli cells. Subsequently, the expression pattern of ZNF185 in mouse testis was determined at different developmental stages. The results demonstrated that the expression of ZNF185 was highest in the testis of 10‑week‑old mice and lowest in 2‑week‑old mice. Furthermore, the role of ZNF185 in Leydig cells of the mouse testis was investigated. Different concentrations of luteinizing hormone (LH) were used to stimulate the Leydig cells and subsequently the expression of ZNF185, and testosterone concentration was detected. The results revealed that LH upregulated the expression of ZNF185 and testosterone secretion, and ZNF185 expression was significantly positively correlated with testosterone secretion. To further validate whether ZNF185 was involved in testosterone secretion, lentiviral‑mediated RNA interference was used to knock down ZNF185 expression in Leydig cells. The results demonstrated that ZNF185 expression and testosterone secretion of Leydig cells were decreased significantly. In addition, the results demonstrated that the knockdown of ZNF185 expression did not significantly affect cell cycle progression or apoptosis. Taken together, the results of the present study revealed that ZNF185 was highly expressed in Leydig cells of the testis and involved in the secretion of testosterone. These results have contributed to the elucidation of the mechanism underlying male reproduction and may provide a novel target for the treatment of infertility, and the development of a contraceptive vaccine.

Klotho, an antiaging protein, can extend the lifespan and modulate cellular responses to inflammation and oxidative stress which can ameliorate chronic kidney diseases (CKD). To investigate the molecular mechanism of Klotho on inflammation in cyclosporine A (CsA) induced nephropathy, the mice were transfected with adenovirus mediated Klotho gene and treated with cyclosporine A (CsA; 30 mg/kg/day) for 4 weeks. Also, primary human renal proximal tubule epithelial cells (RPTECs) were treated with soluble Klotho protein and LPS. The results showed that Ad-klotho significantly reduced serum creatinine (Scr) and blood urea nitrogen (BUN) caused by CsA, and significantly increased creatinine clearance. Tubule interstitial fibrosis score (TIF), renal 8-OHdG excretion, macrophage infiltration and MCP-1 were decreased after Ad-klotho gene transfer. In addition, the overexpression of Klotho led to increase in the expression of PDLIM2, decreased in the amount of NF-kB p65, and inhibited the production of inflammatory cytokines (TNFα, IL-6, IL-12) and iNOS. Accordingly, in vitro results showed, Klotho enhanced PDLIM2 expression and reduced NF-kB p65 expression, while PDLIM2 siRNA could block the inhibitory effects of Klotho on expression of NF-kB p65. Secretion of inflammatory cytokines was also inhibited by Klotho treatment, and PDLIM2 siRNA hindered regulatory effects of Klotho on the cytokines. Real-time PCR and Luciferase assay showed that Klotho markedly increased expression of PDLIM2 mRNA and PDLIM2 reporter activity in a dose-dependent manner. These findings suggest that Klotho can modulate inflammation via PDLIM2/NF-kB p65 pathway in CsA-induced nephropathy., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: LIM and SH3 domain protein 1 (LASP1) has been implicated in several human malignancies and has been shown to predict PSA recurrence in prostate cancer. However, the anti-tumor effect of LASP1 knockdown and the association between LASP1 and the androgen receptor (AR) remains unclear. The aim of this study is to clarify the significance of LASP1 as a target for prostate cancer, and to test the effect of silencing LASP1 in vivo using antisense oligonucleotides (ASO)., Methods: A tissue microarray (TMA) was performed to characterize the differences in LASP1 expression in prostate cancer treated after hormone deprivation therapy. Flow cytometry was used to analyze cell cycle. We designed LASP1 ASO for knockdown of LASP1 in vivo studies., Results: The expression of LASP1 in TMA was increased after androgen ablation and persisted in castration resistant prostate cancer (CRPC). Also in TMA, compared with LNCaP cell, LASP1 expression is elevated in CRPC cell lines (C4-2 and VehA cells). Interestingly, suppression of AR elevated LASP1 expression conversely, AR activation decreased LASP1 expression. Silencing of LASP1 reduced cell growth through G1 arrest which was accompanied by a decrease of cyclin D1. Forced overexpression of LASP1 promoted cell cycle and induced cell growth which was accompanied by an increase of cyclin D1. Systemic administration of LASP1 ASO with athymic mice significantly inhibited tumor growth in CRPC xenografts., Conclusions: These results indicate that LASP1 is negatively regulated by AR at the transcriptional level and promotes tumor growth through induction of cell cycle, ultimately suggesting that LASP1 may be a potential target in prostate cancer treatment. Prostate 77:309-320, 2017. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Background/aims: Cysteine-rich intestinal protein 1 (CRIP1), a member of the LIM/double zinc finger protein family, is abnormally expressed in several tumour types. However, few data are available on the role of CRIP1 in cancer. In the present study, we aimed to investigate the expression profile and functions of CRIP1 in colorectal cancer., Methods: To examine the protein expression level of CRIP1, immunohistochemistry (IHC) was performed on 56 pairs of colon cancer tissue samples. Western blotting was performed to investigate CRIP1 protein expression in four colon cancer cell lines. The endogenous expression of CRIP1 was suppressed using short interfering RNAs (siRNAs). Cell proliferation assays were used to determine whether CRIP1 silencing affected cell proliferation. Flow cytometry analysis was used to detect cell apoptosis. The effects of silencing CRIP1 on cell migration and invasion was detected using the transwell and wound-healing assays., Results: IHC analysis showed that protein level of CRIP1 was significantly higher in tumour tissue samples than in paired non-tumour tissue samples and that the CRIP1 level was higher in metastatic tissue samples than in non-metastatic tissue samples. In addition, protein levels of CRIP1 were higher in highly metastatic colon cancer cell lines than in colon cancer cell lines with low metastasis. Further, CRIP1 silencing had no effect on cell proliferation or apoptosis in SW620 and HT29 cells. CRIP1 silencing suppressed cell migration and invasion obviously in SW620 and HT29 cells., Conclusion: The present study provides new evidence that abnormal expression of CRIP1 might be related to the degree of metastasis in colorectal cancer and that CRIP1 silencing could effectively inhibit migration and invasion during colorectal cancer development. These findings might aid the development of a biomarker for colon cancer prognosis and metastasis, and thus help to treat this common type of cancer., (© 2017 The Author(s). Published by S. Karger AG, Basel.)

Pulmonary arterial hypertension is a complex disease with multiple etiologic factors. PDLIM5, a member of the Enigma subfamily of PDZ and LIM domain protein family, contains an N-terminal PDZ domain and three LIM domains at its C-terminus. We have previously shown that overexpression of PDLIM5 prevents hypoxia-induced pulmonary hypertension (PH), and deletion of PDLIM5 in smooth muscle cells enhances hypoxia-induced PH in vivo. These results suggest that PDLIM5 may be a novel therapeutic target of PH. In this study, we aim to establish a high-throughput screening platform for PDLIM5-targeted drug discovery. We generated a stable mink lung epithelial cell line (MLEC) containing a transforming growth factor-β/Smad luciferase reporter with lentivirus-mediated suppression of PDLIM5 (MLEC-shPDLIM5) and measured levels of Smad2/3 and pSmad2/3. We found that in MLEC, suppression of PDLIM5 decreased Smad-dependent luciferase activity, Smad3, and pSmad3. We used MLEC-shPDLIM5 and a control cell line (MLEC-shCTL) to screen the Prestwick library (1200 compounds) and identified and validated paclitaxel as a PDLIM5 inhibitor in MLEC. Furthermore, we showed that paclitaxel inhibited Smad2 expression and Smad3 phosphorylation in A549 cells. Our study suggests that this system is robust and suitable for PDLIM5-targeted drug discovery., (© 2016 Society for Laboratory Automation and Screening.)

Background: Although PDLIM2 gene may have a context-dependent role in various human malignancies and can be a potential therapeutic target, only a limited number of in vitro studies addressed the molecular functions of PDLIM2 in prostate cancer. Here, we aimed to explore the role of PDLIM2 and the effect of the PDLIM2 gene suppression on oncogenic phenotypes of human castration-resistant prostate cancer (CRPC)-like cells., Methods: We used human CRPC-like cell lines (PC3, DU145, and C4-2B) for our experiments. Transcription levels of PDLIM2 and relevant genes were measured by real time-PCR and protein expression was analyzed by western blot. Cell viability, proliferation, clonogenic growth, and tumor sphere formation were examined after a specific inhibition of PDLIM2 using RNA interference. Flow cytometry was used to examine apoptotic cell death and cell cycle disturbances. Wound healing and transwell migration assays were performed to investigate the invasion capabilities of CRPC-like cells. Additionally, key oncogenic signaling pathways were examined using western blot. Lastly, we evaluated the in vivo efficacy of PDLIM2 suppression on tumor growth of human CRPC xenografts in mice., Results: We observed a significant enhancement of PDLIM2 expression in human CRPC-like cell lines, while a specific inhibition of PDLIM2 reduced cell viability and proliferation due to apoptotic cell death. Conversely, PDLIM2 overexpression significantly reduced cell proliferation compared to the negative control in androgen-sensitive LNCaP cells. Moreover, PDLIM2 suppression led to a decrease of clonogenic growth and tumor sphere formation in three-dimensional cultures with the G2/M cell cycle arrest in human CRPC-like cells. PDLIM2 inhibition also attenuated cellular migration and invasion capabilities of human CRPC-like cells, and reduced the expression of mesenchymal marker. Among several oncogenic signaling pathways, only the MAPK/ERK signaling cascade was decreased by PDLIM2 inhibition and reciprocally, ERK inhibition down-regulated PDLIM2 expression. Importantly, PDLIM2 inhibition remarkably compromised tumor growth in a human CRPC xenograft model., Conclusion: In summary, the suppression of PDLIM2 significantly reduced such oncogenic phenotypes as proliferation, clonogenicity, invasiveness, and tumor cell growth in human CRPC-like cells both in vitro and in vivo, indicating that PDLIM2 may be considered a novel therapeutic target gene for treating human CRPC., (© 2015 Wiley Periodicals, Inc.)

The multifunctional protein Lmo7 has been implicated in some aspects of myogenesis in mammals. Here we studied the distribution and expression of Lmo7 and the effects of Lmo7 knockdown in primary cultures of chick skeletal muscle cells. Lmo7 was localized within the nuclei of myoblasts and at the perinuclear region of myotubes. Knockdown of Lmo7 using siRNA specific to chick reduces the number and width of myotubes and the number of MyoD positive-myoblasts. Both Wnt3a enriched medium and Bio, activators of the Wnt/beta-catenin pathway, could rescue the effects of the Lmo7 knockdown suggesting a crosstalk between the Wnt/beta-catenin and Lmo7-mediated signaling pathways. Our data shows a role of Lmo7 during the initial events of chick skeletal myogenesis, particularly in myoblast survival., (© 2016 Federation of European Biochemical Societies.)

Four and a half LIM protein 1 (FHL1) has been characterized as a tumor suppressor in various types of tumor. However, the biological function and underlying mechanism of FHL1 in tongue squamous cell carcinoma (TSCC) remain to be elucidated. The present study demonstrated that FHL1 inhibits anchorage‑dependent and ‑independent growth of TSCC cells in vitro and tumor growth in nude mice, as determined by cell proliferation and soft agar assays. Knockdown of FHL1 with FHL1 small interfering RNA (siRNA) promoted tumor growth in nude mice. Mechanistically, flow cytometric analysis showed that knockdown of FHL1 promoted G1/S cell cycle progression. Furthermore, expression of cell cycle‑associated regulators, cyclin D and cyclin E, were detected by western blotting and reverse transcription‑quantitative polymerase chain reaction. Cyclin D and cyclin E were markedly elevated at both the protein and mRNA level in the FHL1 siRNA‑transfected cells. These results suggested that FHL1 has a tumor suppressive role in TSCC and that FHL1 may be a useful target for TSCC gene therapy.

Hypoxia-inducible factors (HIFs) are master regulators of adaptive responses to low oxygen, and their α-subunits are rapidly degraded through the ubiquitination-dependent proteasomal pathway after hydroxylation. Aberrant accumulation or activation of HIFs is closely linked to many types of cancer. However, how hydroxylation of HIFα and its delivery to the ubiquitination machinery are regulated remains unclear. Here we show that Rho-related BTB domain-containing protein 3 (RHOBTB3) directly interacts with the hydroxylase PHD2 to promote HIFα hydroxylation. RHOBTB3 also directly interacts with the von Hippel-Lindau (VHL) protein, a component of the E3 ubiquitin ligase complex, facilitating ubiquitination of HIFα. Remarkably, RHOBTB3 dimerizes with LIMD1, and constructs a RHOBTB3/LIMD1-PHD2-VHL-HIFα complex to effect the maximal degradation of HIFα. Hypoxia reduces the RHOBTB3-centered complex formation, resulting in an accumulation of HIFα. Importantly, the expression level of RHOBTB3 is greatly reduced in human renal carcinomas, and RHOBTB3 deficiency significantly elevates the Warburg effect and accelerates xenograft growth. Our work thus reveals that RHOBTB3 serves as a scaffold to organize a multi-subunit complex that promotes the hydroxylation, ubiquitination and degradation of HIFα.

Epidermal growth factor receptor (EGFR) signaling plays an important role in tumor cell resistance to therapy. In addition to ligand binding, mutual and cooperative interactions of EGFR with integrin cell adhesion receptors critically influence proper downstream signaling through a number of bridging adapter proteins. In the present study, we analyzed the role of two of these adapter proteins, called PINCH1 and Nck2, for cellular radioresistance in combination with EGFR-targeting using the monoclonal antibody cetuximab. siRNA-mediated knockdown of PINCH1 or Nck2 resulted in enhanced radiosensitivity of 3D grown human squamous cell carcinoma cell lines FaDu (head and neck) and A431 (epidermis) comparable with effects seen after cetuximab treatment. Combination of knockdown and cetuximab did not result in additive nor synergistic effects regarding clonogenic radiation survival. Modifications in MAPK, Akt and FAK phosphorylation occurred upon cetuximab treatment as well as PINCH1 or Nck2 depletion. We further found this tumor cell radiosensitization to be due to attenuated repair of DNA double strand breaks and altered Rad50 and Nbs1 expression but without changes in other DNA repair proteins such as ATM, DNA-PK and Mre11. Our data suggest that the adaptor proteins PINCH1 and Nck2 critically contribute to cellular radioresistance and proper EGFR signaling in 3D lrECM grown human squamous cell carcinoma cells. Further investigations are warranted to identify the intracellular signaling network controlled by EGFR, PINCH1 and Nck2.

Growth cone collapse is a critical repulsive response to various guidance cues for axon guidance. Protein kinase C epsilon (PKCε) plays important regulation roles in such responses. Translocation of PKCε from cytoplasm to membrane is crucial to archive its regulatory roles in this process. We previously reported that PDLIM5 could selectively recruit PKCε to its specific substrate in neurons. However, the molecular mechanism of PKCε translocation in the neuronal growth cone collapse remains elusive. Here, we demonstrated that PDLIM5 and PKCε co-existed in the nerve growth cones. By interacting with α-actinin, but not β-actin or β-tubulin, PDLIM5 might contribute to regulation of remodeling of the microfilaments in neurons. Meanwhile, PDLIM5 could also bind to PKCε to form PDLIM5-PKCε complexes in growth cones. In the primary cultured neurons, activation of PKCε by PMA resulted in translocation of both PKCε and PDLIM5 from cytoplasm to the membrane. Knockdown of either PDLIM5 or PKCε rescued the neuron from PMA-induced growth cone collapse. Furthermore, in neurons, application of PDLIM5 shRNA or over-expression of PDLIM5 LIM1-3 mutants reduced the amount of PKCε in the membrane. Together, these results suggest that PDLIM5 acts as a scaffold protein by mediated PKCε translocated to the membrane in PMA-induced growth cone collapse., (Copyright © 2014 Elsevier Inc. All rights reserved.)

As some of the most widely utilised intercellular signalling molecules, transforming growth factor β (TGFβ) superfamily members play critical roles in normal development and become disrupted in human disease. Establishing appropriate levels of TGFβ signalling involves positive and negative feedback, which are coupled and driven by the same signal transduction components (R-Smad transcription factor complexes), but whether and how the regulation of the two can be distinguished are unknown. Genome-wide comparison of published ChIP-seq datasets suggests that LIM domain binding proteins (Ldbs) co-localise with R-Smads at a substantial subset of R-Smad target genes including the locus of inhibitory Smad7 (I-Smad7), which mediates negative feedback for TGFβ signalling. We present evidence suggesting that zebrafish Ldb2a binds and directly activates the I-Smad7 gene, whereas it binds and represses the ligand gene, Squint (Sqt), which drives positive feedback. Thus, the fine tuning of TGFβ signalling derives from positive and negative control by Ldb2a. Expression of ldb2a is itself activated by TGFβ signals, suggesting potential feed-forward loops that might delay the negative input of Ldb2a to the positive feedback, as well as the positive input of Ldb2a to the negative feedback. In this way, precise gene expression control by Ldb2a enables an initial build-up of signalling via a fully active positive feedback in the absence of buffering by the negative feedback. In Ldb2a-deficient zebrafish embryos, homeostasis of TGFβ signalling is perturbed and signalling is stably enhanced, giving rise to excess mesoderm and endoderm, an effect that can be rescued by reducing signalling by the TGFβ family members, Nodal and BMP. Thus, Ldb2a is critical to the homeostatic control of TGFβ signalling and thereby embryonic patterning.

The TES gene was frequently lost in breast cancer, which could inhibit tumor invasion and the formation of distant metastasis. However, the underlying mechanisms remain unknown yet. In the present study, we aimed to investigate how TES was silenced and its roles in EMT--the key step for tumor metastasis. Real-time polymerase chain reaction (PCR) and Western blot were used to detect the mRNA and protein expression of target genes; the status of TES promoter was determined by methylation-specific PCR and subsequently, DNA sequencing. Overexpression or downregulation of TES was achieved by pcDNA3.1-TES or shRNA-TES transfection. Cellular adhesion and migration were investigated by the adhesion and Transwell assays. Morphological changes of breast cancer cells were observed under the optical microscope. The Rho A activity was measured using a commercial kit, and its roles in TES-manipulated EMT were determined by real-time PCR and Western blot. The 42.3% (33/78) breast cancer tissues presented hypermethylation of the TES gene, whereas only 2 (2.6%) non-malignant cases were hypermethylated (P<0.001). Moreover, TES hypermethylation was significantly correlated with larger tumor diameter (P=0.03) and lympho node metastasis (P=0.024). In primary cultured breast cancer cells, the demethylation treatment using 5-aza-dC notably restored the expression of TES. In vitro, overexpression of TES enhanced cellular adhesion inhibited migration and suppressed EMT, while downregulation of TES impaired cellular adhesion, promoted migration, and enhanced EMT. TES overexpression also activated the Rho A signal, which is a critical factor for the effects of TES on the EMT procedure. We firstly proved that frequent loss of TES in breast cancer was caused by promoter hypermethylation, which was correlated with poor prognosis. In vitro, TES enhanced cellular adhesion, suppressed tumor migration, and inhibited EMT. Moreover, the Rho A pathway was critical for the effects of TES on EMT, which can be blocked by the Rho A inhibitor. Therefore, we propose restoration of TES as a potent strategy for breast cancer therapy.

Metastatic colonization of distant organs underpins the majority of human-cancer-related deaths, including deaths from head and neck squamous cell carcinoma (HNSCC). We report that miR-203, a miRNA that triggers differentiation in multilayered epithelia, inhibits multiple postextravasation events during HNSCC lung metastasis. Inducible reactivation of miR-203 in already established lung metastases reduces the overall metastatic burden. Using an integrated approach, we reveal that miR-203 inhibits metastasis independently of its effects on differentiation. In vivo genetic reconstitution experiments show that miR-203 inhibits lung metastasis by suppressing the prometastatic activities of three factors involved in cytoskeletal dynamics (LASP1), extracellular matrix remodeling (SPARC), and cell metabolism (NUAK1). Expression of miR-203 and its downstream effectors correlates with HNSCC overall survival outcomes, indicating the therapeutic potential of targeting this signaling axis., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

In mammals, formation of the auditory sensory organ (the organ of Corti) is restricted to a specialized area of the cochlea. However, the molecular mechanisms limiting sensory formation to this discrete region in the ventral cochlear duct are not well understood, nor is it known whether other regions of the cochlea have the competence to form the organ of Corti. Here we identify LMO4, a LIM-domain-only nuclear protein, as a negative regulator of sensory organ formation in the cochlea. Inactivation of Lmo4 in mice leads to an ectopic organ of Corti (eOC) located in the lateral cochlea. The eOC retains the features of the native organ, including inner and outer hair cells, supporting cells, and other nonsensory specialized cell types. However, the eOC shows an orientation opposite to the native organ, such that the eOC appears as a mirror-image duplication to the native organ of Corti. These data demonstrate a novel sensory competent region in the lateral cochlear duct that is regulated by LMO4 and may be amenable to therapeutic manipulation., (Copyright © 2014 the authors 0270-6474/14/3410072-06$15.00/0.)

PKD (protein kinase D) is a serine/threonine kinase implicated in multiple cardiac roles, including the phosphorylation of the class II HDAC5 (histone deacetylase isoform 5) and thereby de-repression of MEF2 (myocyte enhancer factor 2) transcription factor activity. In the present study we identify FHL1 (four-and-a-half LIM domains protein 1) and FHL2 as novel binding partners for PKD in cardiac myocytes. This was confirmed by pull-down assays using recombinant GST-fused proteins and heterologously or endogenously expressed PKD in adult rat ventricular myocytes or NRVMs (neonatal rat ventricular myocytes) respectively, and by co-immunoprecipitation of FHL1 and FHL2 with GFP-PKD1 fusion protein expressed in NRVMs. In vitro kinase assays showed that neither FHL1 nor FHL2 is a PKD1 substrate. Selective knockdown of FHL1 expression in NRVMs significantly inhibited PKD activation and HDAC5 phosphorylation in response to endothelin 1, but not to the α₁-adrenoceptor agonist phenylephrine. In contrast, selective knockdown of FHL2 expression caused a significant reduction in PKD activation and HDAC5 phosphorylation in response to both stimuli. Interestingly, neither intervention affected MEF2 activation by endothelin 1 or phenylephrine. We conclude that FHL1 and FHL2 are novel cardiac PKD partners, which differentially facilitate PKD activation and HDAC5 phosphorylation by distinct neurohormonal stimuli, but are unlikely to regulate MEF2-driven transcriptional reprogramming.

Background and Purpose: 4-Phenylbutyric acid (4-PBA) is a chemical chaperone that eliminates the accumulation of unfolded proteins in the endoplasmic reticulum (ER). However, its chaperoning ability is often weak and unable to attenuate the unfolded protein response (UPR) in vitro or in vivo. To develop more potent chemical chaperones, we synthesized six analogues of 4-PBA and evaluated their pharmacological actions on the UPR., Experimental Approach: NRK-52E cells were treated with ER stress inducers (tunicamycin or thapsigargin) in the presence of each of the 4-PBA analogues; the suppressive effects of these analogues on the UPR were assessed using selective indicators for individual UPR pathways., Key Results: 2-POAA-OMe, 2-POAA-NO2 and 2-NOAA, but not others, suppressed the induction of ER stress markers GRP78 and CHOP. This suppressive effect was more potent than that of 4-PBA. Of the three major UPR branches, the IRE1 and ATF6 pathways were markedly blocked by these compounds, as indicated by suppression of XBP1 splicing, inhibition of UPRE and ERSE activation, and inhibition of JNK phosphorylation. Unexpectedly, however, these agents did not inhibit phosphorylation of PERK and eIF2α triggered by ER stress. These compounds dose-dependently inhibited the early activation of NF-κB in ER stress-exposed cells. 2-POAA-OMe and 2-POAA-NO2 also inhibited ER stress-induced phosphorylation of Akt., Conclusion and Implications: The 4-PBA analogues 2-POAA-OMe, 2-POAA-NO2 and 2-NOAA strongly inhibited activation of the IRE1 and ATF6 pathways and downstream pathogenic targets, including NF-κB and Akt, in ER stress-exposed cells. These compounds may be useful for therapeutic intervention in ER stress-related pathological conditions., (© 2013 The British Pharmacological Society.)

Nephrin, a structural molecule, is also a signaling molecule after phosphorylation. Inhibition of nephrin phosphorylation is correlated with podocyte injury. The PINCH-1-ILK-α-parvin (PIP) complex plays a crucial role in cell adhesion and cytoskeleton formation. We hypothesized that nephrin phosphorylation influenced cytoskeleton and cell adhesion in podocytes by regulating the PIP complex. The nephrin phosphorylation, PIP complex formation, and F-actin in Wistar rats intraperitoneally injected with puromycin aminonucleoside were gradually decreased but increased with time, coinciding with the recovery from glomerular/podocyte injury and proteinuria. In cultured podocytes, PIP complex knockdown resulted in cytoskeleton reorganization and decreased cell adhesion and spreading. Nephrin and its phosphorylation were unaffected after PIP complex knockdown. Furthermore, inhibition of nephrin phosphorylation suppressed PIP complex expression, disorganized podocyte cytoskeleton, and decreased cell adhesion and spreading. These findings indicate that alterations in nephrin phosphorylation disorganize podocyte cytoskeleton and decrease cell adhesion through a PIP complex-dependent mechanism.

LIM and SH3 protein 1 (LASP-1) plays a significant role in the formation of several malignant tumours. However, the biological and clinical significances of LASP-1 in hepatocellular carcinoma (HCC) remain largely unknown. Using immunohistochemistry, we analysed LASP-1 expression in 144 clinicopathologically characterised HCC cases. Using gene and transfection and RNA interference, we investigated the effects of LASP-1 overexpression and depletion on tumour cellular behaviour in vitro. LASP-1 expression was detected in not only cytoplasm and but also nucleus of HCC and liver cells. The positive rates of both cytosolic and nuclear LASP-1 expression in HCC were higher than adjacent non-tumourous tissues. Statistical analysis showed that heterogeneous LASP-1 expression is associated with hepatitis B surface antigen (HBsAg) and serum alpha-fetoprotein (AFP) level of HCC patients. A significant trend was identified between cytosolic LASP-1 overexpression in HCC and worsening clinical prognosis. Multivariate survival analysis showed that cytosolic LASP-1 expression was recognised as an independent prognostic factor of patient's survival. In vitro study showed LASP-1 promoted cell proliferation and migration, and resulted in aggressive phenotypes of cancer cells. LASP-1 is a valuable marker of HCC progression. High cytosolic LASP-1 expression is associated with poor overall survival in HCC patients., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Pinch-1, a widely expressed focal adhesion protein, has been demonstrated to be up-regulated in multiple solid tumor-associated stromal cells, particularly at invasive edges. It was supposed that Pinch-1 was intimately associated with development and progression of tumors. The expression of Pinch-1 in hematopoietic microenvironment in patients with leukemia remains unclear. This study focused on the expression of Pinch-1 in bone marrow stromal cells (BMSCs) from leukemia patients and its possible effect. BMSC was isolated and cultured from bone marrow in leukemia patients and normal healthy donors. RT-PCR and Western blot analysis were performed to determine Pinch-1 mRNA and protein level in BMSC, respectively. Lentiviral vector containing Pinch-1 siRNA was constructed, and the recombinant lentivirus particle was packaged in 293 cells. Effectiveness of Pinch-1 siRNA was determined by Western blot. The proliferation, apoptosis and motility of leukemia BMSC subjected to Pinch-1 knockdown using siRNA were tested by flow cytometry, TUNEL assay and Transwell system, respectively. Pinch-1 mRNA and protein were significantly up-regulated in ALL and AML BMSC compared to normal BMSC (p<0.01). Although there was no difference in Pinch-1 mRNA between ALL and AML BMSC, cellular levels of Pinch-1 protein in ALL BMSC were significantly higher than that in AML BMSC (p<0.01). Overexpressed Pinch-1 was significantly reduced in leukemia BMSC transfected with Pinch-1 siRNA evidenced by Western blot. Flow cytometry analysis showed that the percentage of cells in S+G2 phases in leukemia BMSC transfected with Pinch-1 siRNA was significantly lower than control (p<0.01). The percentage of apoptotic cells in leukemia BMSC transfected with Pinch-1 siRNA was 19.8±1.0%, significantly higher than controls (p<0.01). The number of leukemia BMSC transfected with Pinch-1 siRNA that migrated to the lower chamber after culturing for 24 h was 8.4±1.1 per field, significantly lower than controls (p<0.01). Pinch-1 mRNA and protein in leukemia BMSC were up-regulated drastically compared with BMSC from healthy donors. Leukemia BMSC displayed hypoproliferation, decreased migration and increased apoptosis after transfecting Pinch-1 siRNA.

Vascular endothelial growth factor (VEGF) plays an important role in many disease states, including ischemia, chronic and acute inflammation, and pathologies associated with angiogenesis such as tumors and wounds. A number of factors regulate VEGF promoter activity and VEGF expression such as four and a half LIM domains 1 (FHL1) and Smad4. FHL1 belongs to a family of LIM-only proteins that regulate gene transcription, cell proliferation, differentiation and apoptosis. Smad4 is a tumor suppressor gene, initially identified as deleted in pancreatic carcinoma locus 4 (DPC4). The aim of this study was to determine whether FHL1 and Smad4 inhibited VEGF signaling. HepG2 cells were transfected with the VEGF-Luc reporter, Smad4 and FHL1 or Smad4 and FHL1 siRNA. Results showed that the overexpression of FHL1 and Smad4 synergistically inhibited the promoter activity, mRNA expression and secretion of VEGF, whereas knockdown of endogenous Smad4 and FHL1 had opposite effects. Moreover, the reduction of endogenous Smad4 eliminated FHL1-mediated inhibition of the VEGF promoter activity. In conclusion, a cooperative regulation of VEGF signaling by FHL1 and Smad4 was evidenced, which may provide a novel regulation mechanism underlying cancer development and progression.

Mice treated with MK-801, a non-competitive antagonist of the N-methyl-d-aspartic (NMDA) acid receptor, are important animal models for schizophrenia studies. In the present study, we compared protein expression levels in the hippocampus of mice treated with MK-801 (0.6 mg/kg) or saline once daily for 7 days. Changes in the proteome were detected by two-dimensional electrophoresis, and the six proteins exhibiting differential expression were identified by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. Down-regulation of one of these proteins, Lasp1 (LIM and SH3 protein 1), in MK-801-treated mice was confirmed by western blotting and immunohistochemical analyses. Lasp1 is a multidomain protein that may recruit signaling molecules to the actin-based cytoskeleton and is known to concentrate in synaptic sites of hippocampal neurons. We next investigated whether polymorphisms in the human LASP1 gene were associated with schizophrenia in the Korean population. A single-nucleotide polymorphism in the LASP1 gene promoter region was associated with schizophrenia susceptibility. Our results suggest that LASP1 might be associated with NMDA receptor antagonism and schizophrenia susceptibility and, thus, might be involved in the pathophysiology of schizophrenia., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Four and a half LIM domain (FHL) proteins belong to a family of LIM-only proteins that have been implicated in the development and progression of various types of cancers. However, the role of FHL proteins in tumor angiogenesis remains to be elucidated. Herein, we demonstrate that FHL1-3 decrease the promoter activity and expression of vascular endothelial growth factor (VEGF), the key regulator of angiogenesis in cancer growth and progression as well as an important target gene of the transcription factor hypoxia-inducible factor 1 (HIF1α/HIF1β). FHL1-3 interacted with HIF1α both in vitro and in vivo. A single LIM domain of FHL1 was sufficient for its interaction with HIF1α. FHL1 interacted with the HIF1α region containing basic helix-loop-helix (bHLH) motif and PER-ARNT-SIM domain, both of which aid in dimerization with HIF1β and DNA binding. FHL1-3 inhibited HIF1 transcriptional activity and HIF1-mediated VEGF expression in a hypoxia-independent manner. Moreover, FHL1 blocked HIF1α-HIF1β heterodimerization and HIF1α recruitment to the VEGF promoter. These data suggest that FHL proteins are involved in negative regulation of VEGF possibly by interfering with the dimerization and DNA binding of HIF1 subunits and may play an important role in tumor angiogenesis., (Copyright © 2012 International Union of Biochemistry and Molecular Biology, Inc.)

Transforming growth factor β (TGF-β)-stimulated epithelial-mesenchymal transition (EMT) is an important developmental process that has also been implicated in increased cell invasion and metastatic potential of cancer cells. Expression of the focal adhesion protein Hic-5 has been shown to be up-regulated in epithelial cells in response to TGF-β. Herein, we demonstrate that TGF-β-induced Hic-5 up-regulation or ectopic expression of Hic-5 in normal MCF10A cells promoted increased extracellular matrix degradation and invasion through the formation of invadopodia. Hic-5 was tyrosine phosphorylated in an Src-dependent manner after TGF-β stimulation, and inhibition of Src activity or overexpression of a Y38/60F nonphosphorylatable mutant of Hic-5 inhibited matrix degradation and invasion. RhoC, but not RhoA, was also required for TGF-β- and Hic-5-induced matrix degradation. Hic-5 also induced matrix degradation, cell migration, and invasion in the absence of TGF-β via Rac1 regulation of p38 MAPK. These data identify Hic-5 as a critical mediator of TGF-β-stimulated invadopodia formation, cell migration, and invasion.

Coronary artery disease (CAD) is the leading cause of death worldwide. It has been established that internal mammary arteries (IMA) are resistant to the development of atherosclerosis, whereas left anterior descending (LAD) coronary arteries are athero-prone. The contrasting properties of these two arteries provide an innovative strategy to identify the genes that play important roles in the development of atherosclerosis. We carried out microarray analysis to identify genes differentially expressed between IMA and LAD. Twenty-nine genes showed significant differences in their expression levels between IMA and LAD, which included the TES gene encoding Testin. The role of TES in the cardiovascular system is unknown. Here we show that TES is involved in endothelial cell (EC) functions relevant to atherosclerosis. Western blot analysis showed higher TES expression in IMA than in LAD. Reverse transcription polymerase chain reaction and western blot analyses showed that TES was consistently and markedly down-regulated by more than 6-fold at both mRNA and protein levels in patients with CAD compared with controls without CAD (P= 0.000049). The data suggest that reduced TES expression is associated with the development of CAD. Knockdown of TES expression by small-interfering RNA promoted oxidized-LDL-mediated monocyte adhesion to ECs, EC migration and the transendothelial migration of monocytes, while the over-expression of TES in ECs blunted these processes. These results demonstrate association between reduced TES expression and CAD, establish a novel role for TES in EC functions and raise the possibility that reduced TES expression increases susceptibility to the development of CAD.

The Drosophila melanogaster MICAL protein is essential for the neuronal growth cone machinery that functions through plexin- and semaphorin-mediated axonal signaling. Drosophila MICAL is also involved in regulating myofilament organization and synaptic structures, and serves as an actin disassembly factor downstream of plexin-mediated axonal repulsion. In mammalian cells there are three known isoforms, MICAL1, MICAL2 and MICAL3, as well as the MICAL-like proteins MICAL-L1 and MICAL-L2, but little is known of their function, and information comes almost exclusively from neural cells. In this study we show that in non-neural cells human MICALs are required for normal actin organization, and all three MICALs regulate actin stress fibers. Moreover, we provide evidence that the generation of reactive oxygen species by MICAL proteins is crucial for their actin-regulatory function. However, although MICAL1 is auto-inhibited by its C-terminal coiled-coil region, MICAL2 remains constitutively active and affects stress fibers. These data suggest differential but complementary roles for MICAL1 and MICAL2 in actin microfilament regulation.

Hydrogen peroxide-inducible clone-5 (Hic-5) is a focal adhesion scaffold protein primarily expressed in vascular and visceral smooth muscle cells. We recently generated mice lacking Hic-5, which grew with no apparent abnormality (Kim-Kaneyama J, et al. J Mol Cell Cardiol. 2011;50(1):77-86). However, we discovered that recovery of arterial media following vascular injury is delayed significantly in Hic-5 knockout mice consequent to enhanced apoptosis of cultured vascular smooth muscle cells after mechanical stress; thus, Hic-5 is regarded as a novel factor in vascular remodeling. The Hic-5 gene is also induced by transforming growth factor-β, a well-known accelerator in fibrosis. Hic-5 involvement in various fibrotic disorders, e.g., scar formation, keloid formation and glomerulosclerosis, has been proposed. siRNA silencing of Hic-5 in a breast cancer cell line reduces its invasiveness; moreover, Hic-5 serves as a steroid hormone co-activator and likely participates in endometriosis and prostate cancer. Thus, functional characterization of Hic-5 in various pathophysiological conditions may afford novel mechanistic insights into a wide variety of diseases.

Monomeric actin regulates gene expression through serum response factor (SRF) by inhibiting its transcriptional coactivator myocardin-related transcription factor (MAL/MRTF). Many affected genes encode cytoskeletal components. We have analysed the migratory effects of actin-MAL signalling and of new target genes in non-invasive highly adherent cells. Expression of active MAL impaired migration of both fibroblasts and epithelial cells, whereas dominant-negative constructs and partial knockdown of MAL/MRTF enhanced motility. Knockdown of three newly characterised G-actin-regulated MAL targets, integrin α5, plakophilin 2 (Pkp2) and FHL1, enhanced cell migration. All three were upregulated by external stimulation through actin-MAL-SRF signalling, and MAL and SRF were inducibly recruited to cis-regulatory elements of the integrin α5 and Pkp2 genes. Finally, the reduced migration of epithelial cells stably expressing MAL was partially reversed by knockdown of Pkp2 and FHL1. We conclude that the actin-MAL pathway promotes adhesive gene expression, including integrin α5, Pkp2 and FHL1, and that this is anti-motile for non-invasive cells harbouring high basal activity.

Granuloma formation is an important host defense mechanism against intracellular bacteria; however, uncontrolled granulomatous inflammation is pathologic. T helper 17 (TH17) cells are thought to have a pathogenic role in autoimmune and inflammatory diseases, including in granulomas. Here, we report that the PDZ-LIM domain protein PDLIM2 inhibited TH17 cell development and granulomatous responses by acting as a nuclear ubiquitin E3 ligase that targeted signal transducer and activator of transcription 3 (STAT3), a transcription factor critical for the commitment of naïve CD4+ T cells to the TH17 lineage. PDLIM2 promoted the polyubiquitination and proteasomal degradation of STAT3, thereby disrupting STAT3-mediated gene activation. Deficiency in PDLIM2 resulted in the accumulation of STAT3 in the nucleus, enhanced the extent of TH17 cell differentiation, and exacerbated granuloma formation. This study delineates an essential role for PDLIM2 in inhibiting TH17 cell-mediated inflammatory responses by suppressing STAT3 signaling and provides a potential therapeutic target for the treatment of autoimmune diseases.

Mical-1 is a novel F-actin-disassembly factor that is critical in actin reorganization. It provides a molecular conduit through which actin reorganizes-a hallmark of cell morphological changes, including axon navigation. However, whether Mical-1 is involved in the epileptogenesis remains unknown. Here, we investigate Mical-1 expression pattern in patients with intractable temporal lobe epilepsy (TLE) and pilocarpine-induced rat model. We used double-labeled immunoflurescence, immunohistochemistry, and Western blotting to assess the location and expression of Mical-1 in temporal neocortex of patients with intractable TLE, and the expression pattern of Mical-1 at different time point in the hippocampus and temporal lobe cortex of the pilocarpine-induced rat model. Double-labeled immunofluorescence showed that Mical-1 was coexpressed with neuron-specific enolase (NSE) in the cytoplasm of neurons in temporal neocortex of patients with TLE and hippocampus of rat model. Faint and scattered immunoreactivity for Mical-1 in the neuron of temporal neocortex in TLE group, but strong immunoreactivity for Mical-1 was shown in control subjects. To quantitatively evaluate the Mical-1 immunoreactivity, we measured the mean optical density (OD) of Mical-1. In the hippocampus of pilocarpine-induced rat model, the OD values transient increased at 6 h after seizure then decreased from 1 day to 14 days, and returned to a subnormal level at 60 days. The lowest level of Mical-1 expression occurred at 14 days after seizure in the hippocampus. In the temporal lobe cortex of rat model, the OD values decreased at all time point after kindling compared to the normal group. Furthermore, our Western blot analysis confirmed these expression patterns of Mical-1 from latent stage to chronic stage. Our results indicate that in patients with TLE and pilocarpine-induced rat model, the expression of Mical-1 were followed a downtrend from the latent stage to chronic stage after seizure evoke. Thus, as an effect factor participated in F-actin disassemble, Mical-1 may associate with inner pathophysiological modulation in epilepsy., (Copyright © 2011 Wiley-Liss, Inc.)