Overexpression Of Profilin Research Articles

Profilin1 Promotes Renal Tubular Epithelial Cell Apoptosis in Diabetic Nephropathy Through the Hedgehog Signaling Pathway.

Profilin-1 (PFN1) regulates the dynamic balance of actin and plays an important role in cell functions as a hub protein in signaling molecule interaction networks. Dysregulation of PFN1 is related to pathologic kidney diseases. Diabetic nephropathy (DN) was recently reported as an inflammatory disorder, however, the molecular mechanisms of PFN1 in DN remain unclear. Therefore, the present study was conducted to explore the molecular and bioinformatic characteristics of PFN1 in DN. Bioinformatics analyses were performed on the chip of database in DN kidney tissues. A cellular model of DN was established in human renal tubular epithelial cells (HK-2) induced by high glucose. The PFN1 gene was overexpressed or knocked-down to investigate its function in DN. Flow cytometry was used to detect cell proliferation and apoptosis. PFN1 and proteins in the related signaling pathways were evaluated by Western blotting. The expression of PFN1 was significantly increased in DN kidney tissues (P < 0.001) and was correlated with a high apoptosis-associated score (Pearson's correlation = 0.664) and cellular senescence-associated score (Pearson's correlation = 0.703). PFN1 protein was mainly located in cytoplasm. Overexpression of PFN1 promoted apoptosis and blocked the proliferation of HK-2 cells treated with high levels of glucose. Knockdown of PFN1 led to the opposite effects. Additionally, we found that PFN1 was correlated with the inactivation of the Hedgehog signaling pathway in HK-2 cells treated with high levels of glucose. PFN1 might play an integral role in the regulation of cell proliferation and apoptosis during DN development by activating the Hedgehog signaling pathway. This study provided molecular and bioinformatic characterizations of PFN1, and contributed to the understanding of the molecular mechanisms leading to DN.

Profilin1 Regulates Trophoblast Invasion and Macrophage Differentiation

Unexplained recurrent spontaneous abortion (URSA) has been associated with the dysfunction of trophoblasts and decidual macrophages. Current evidence suggests that profilin1 (PFN1) plays an important role in many biological processes. However, little is known about whether PFN1 is related to URSA. Herein, the location of PFN1 was detected by immunohistochemistry, and the level of PFN1 was detected by quantitative real-time PCR, Western blot analysis, and immunohistochemistry. The proliferation of trophoblasts was detected by CCK8 and 5-ethynyl-2'-deoxyuridine assays, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assays were used to detect apoptosis of trophoblasts. The migration and invasion ability of trophoblasts was assessed by using the wound-healing test and transwell test. Polarization of macrophages was detected in macrophages cultured in trophoblast conditioned medium. PFN1 expression was observed in cytotrophoblasts, syncytiotrophoblasts, and extravillous trophoblasts and was decreased in the villous tissue of patients with URSA. The migration and invasion ability and cell viability of trophoblastic cell lines that underwent PFN1 knockdown significantly decreased, and apoptosis increased. Opposite findings were observed after the overexpression of PFN1 in trophoblastic cells. In addition, PFN1 could regulate trophoblast function through phosphatidylinositol 3-kinase/AKT signal transduction rather than mitogen-activated proteinkinasesignaling pathways. Finally, knockdown of PFN1 in trophoblasts promoted tumor necrosis factor-α secretion to induce macrophage polarization to M1 phenotype, mediated by the NF-κB signaling pathway. These findings indicate that PFN1 has a broad therapeutic potential for patients with URSA.

Profilin 1 Induces Tumor Metastasis by Promoting Microvesicle Secretion Through the ROCK 1/p-MLC Pathway in Non-Small Cell Lung Cancer.

Profilin 1 (PFN1), an actin-binding protein, plays contrasting roles in the metastasis of several cancers; however, its role in non-small cell lung cancer (NSCLC) metastasis remains unclear. Here, PFN1 expression was upregulated in metastatic NSCLC tissues. PFN1 overexpression significantly promotes NSCLC metastasis in vitro and in vivo. Proteomics analysis revealed PFN1 involvment in microvesicles (MVs) secretion. In vitro experiments confirmed that PFN1 overexpression increased secretion of MVs. MVs are important mediators of metastasis. Here, we show an increased abundance of MVs in the sera of patients with metastatic NSCLC compared to that in the sera of patients with non-metastatic NSCLC. Both in vitro and in vivo experiments revealed that PFN1 could increase MV secretion, and MVs derived from PFN1-overexpressing cells markedly promoted NSCLC metastasis. We then elucidated the mechanisms underlying PFN1-mediated regulation of MVs and found that PFN1 could interact with ROCK1 and enhance its kinase activity to promote myosin light chain (MLC) phosphorylation for MV secretion. Inhibition of ROCK1 decreased MV secretion and partially reversed the PFN1-induced promotion of NSCLC metastasis. Collectively, these findings show that PFN1 regulates MV secretion to promote NSCLC metastasis. PFN1 and MVs represent potential predictors or therapeutic targets for NSCLC metastasis.

P68.10 PFN1 Induces Tumor Metastasis Through Promoting Secretion of Microvesicles By ROCK I/p-MLC Pathway in Non-Small Cell Lung Cancer

Profilin1(PFN1) plays confusing roles in metastasis of different cancers, here we explored the roles of PFN1 in metastasis of non-small cell lung cancer (NSCLC). Considering that PFN1 is an important mediator of membrane trafficking and microvesicles(MVs) have already been proved closely associated with tumor metastasis, we further explored relationship of PFN1 and MVs in NSCLC metastasis and the underlying mechanisms. We first detect PFN1 and p-MLC expression in tissue microarray. Then we extracted MVs of NSCLC patients’ serums by continuous differential centrifugation, quantified MVs by WB, electron microscopy and spectral flow cytometry. And PFN1 was overexpression/ knockdown to study the effect of MVs secretion and cell migration. Protein interaction was tested by CO-IP. ROCK kinase assay was used to detect ROCK kinase activity. For in vivo experiment, intracardiac injection of H1299 cell lines were used to establish metastatic tumor model for in vivo assay. PFN1 were highly expressed in late stage NSCLC tissues, and the expression of PFN1 is positively correlated with p-MLC in IHC. Overexpression of PFN1 promotes MVs secretion and metastasis in vitro. Further investigation found that PFN1 could interact with ROCK I and affect the activity of ROCK I to phosphorylate MLC, which could regulate the secretion of MVs. Inhibition of ROCK I activity by Y27632 partially reverse PFN1’ s promotion of MVs secretion and NSCLC metastasis in vivo and in vitro. PFN1 affects NSCLC metastasis via promoting MVs’ secretion, which provided us a new insight into PFN1’s roles in cancer metastasis and a new target of therapy in NSCLC metastasis.

Mechanism of action of Profilin-1 and Fibulin-3 in vascular remodeling in hypertensive rats.

This paper aims to investigate the expressions of Fibulin-3 and Profilin-1 in vascular remodeling and the relationship between the two factors and vascular remodeling in hypertensive rats. 45 spontaneously hypertensive rats (SHR) and 15 healthy Wistar Kyoto (WKY) rats were collected. The 45 SHR were randomly divided into group A, group B, and group C. Group A was injected with Profilin-1 overexpression of adenoviral vector of pAd-Profilin-1-RES-EGFP; group B was injected with recombinant Fibulin3 protein solution; and group C was injected with normal saline. The rats in the control group were normally raised. All rats were anesthetized and dissected, and the thoracic aorta of rats was taken out at T0 (8 weeks old), T1 (12 weeks old), T2 (16 weeks old). The expressions of Fibulin-3 and Profilin-1 protein in the thoracic aorta were analyzed by Western blot. The overexpression of Profilin-1 and Fibulin-3 protein, blood pressure, and body weight were compared. The expression level and systolic blood pressure of Profilin-1 protein of rats in group A were significantly higher than those in the other two groups (p<0.05). The expression level of Fibulin-3 protein of rats in group B at T2 was significantly higher than that in the other two groups (p<0.05). The thickness of vascular wall in the control group and group C at T1 and T2 was significantly lower than that of group A and group B. The vascular wall/cavity ratio of rats in group A, B, C was significantly higher than that in the control group at T1 and T2 (p<0.001). The changes in Profilin-1 and Fibulin-3 levels may affect the occurrence and development of vascular remodeling in hypertension. Therefore, Profilin-1 and Fibulin-3 can be used as sensitive detection indices for hypertension vascular remodeling.

Actin filaments are dispensable for bulk autophagy in plants

ABSTRACTActin filament, also known as microfilament, is one of two major cytoskeletal elements in plants and plays important roles in various biological processes. Like in animal cells, actin filaments have been thought to participate in autophagy in plants. However, surprisingly, in this study we found that actin filaments are dispensable for the occurrence of autophagy in plants. Disruption of actin filaments by short term treatment with actin polymerization inhibitors, cytochalasin D and latrunculin B, or transient overexpression of Profilin 3 in Nicotiana benthamiana had no effect on basal autophagy as well as the upregulation of nocturnal autophagy and salt stress-induced autophagy. Furthermore, anti-microfilament drug treatment affected neither basal nor salt stress-induced autophagy in Arabidopsis. In addition, prolonged perturbation of actin filaments by silencing Actin7 or 24-h treatment with microfilament-disrupting agents in N. benthamiana caused endoplasmic reticulum (ER) disorganization and subsequent degradation via autophagy involving ATG2, 3, 5, 6 and 7. Our findings reveal that, unlike mammalian cells, actin filaments are unnecessary for bulk autophagy in plants.Abbreviations: ATG: autophagy-related; CD: cytochalasin D; Cvt pathway: cytoplasm to vacuole targeting pathway; DMSO: dimethyl sulfoxide; ER: endoplasmic reticulum; LatB: latrunculin B; Nb: Nicotiana benthamiana; PAS: phagophore assembly site; PRF3: Profilin 3; RER: rough ER; SER: smooth ER; TEM: transmission electron microscopy; TRV: Tobacco rattle virus; VIGS: virus-induced gene silencing; wpi: weeks post-agroinfiltration

Profilin 1, negatively regulated by microRNA-19a-3p, serves as a tumor suppressor in human hepatocellular carcinoma

Profilin 1 (PFN1) is a critical actin-regulatory protein; however, its functional role in hepatocellular carcinoma (HCC) progression remains to be further elucidated. In the present study, we observed that the expression levels of PFN1 were significantly decreased in HCC tissues and cell lines. Low PFN1 expression was significantly correlated with aggressive clinicopathological characteristics and poor prognosis of HCC patients. Further in vitro experiments demonstrated that overexpression of PFN1 remarkably inhibited the proliferation, migration, invasion and EMT of HCC cells. Moreover, we also found that PFN1 was a direct target gene of miR-19a-3p, and in HCC tissues, and there was a significantly inverse correlation between PFN1 mRNA and miR-19a-3p expression. Collectively, our results showed that PFN1 functions as a tumor suppressor in HCC, and might serve as a diagnostic and therapeutic target for HCC patients.

The effect of piperlongumine on endothelial and lung adenocarcinoma cells with regulated expression of profilin-1.

PurposeThe aim of the study was to evaluate the effect of piperlongumine (2 and 4 µM) on endothelial EA.hy926 and lung adenocarcinoma A549 cells with regulated expression of profilin-1 (PFN1).Material and methodsThe cytotoxicity of alkaloid was evaluated by MTT assay, while cell death was assessed using double staining with annexin V and propidium iodide. Subsequently, the level of PFN1 1) upregulation in EA.hy926 endothelial cells and 2) downregulation in A549 lung adenocarcinoma cells. The next step was the analysis of the effect of PFN1 manipulation on cytoskeletal proteins.ResultsThe results showed that piperlongumine may inhibit proliferation of EA.hy926 and A549 cell lines and also induce cell death in a dose-dependent manner. Furthermore, endothelial cells with PFN1 overexpression showed lower sensitivity to alkaloid and strengthening of cell–cell interactions. In the case of A549 cells, loss of PFN1 expression resulted in a lower percentage of early apoptotic cells, reorganization of F-actin and vimentin network, and reduction of migratory potential.ConclusionWe suggest that upregulation of PFN1 in endothelial cell line may stabilize the cell junctions. In turn, PFN1 downregulation in A549 cells probably suppresses cell migration and sensitizes cells to anticancer agents.

Profilin 1 induces drug resistance through Beclin1 complex-mediated autophagy in multiple myeloma.

Autophagy plays an important role in multiple myeloma (MM) for homeostasis, survival and drug resistance, but which genes participate in this process is unclear. We identified several cytoskeleton genes upregulated in MM patients by gene expression profiling (GEP) datasets; in particular, patients with high profilin 1 (PFN1) expression had poor prognosis in MM. In vitro, overexpressed PFN1 promotes proliferation and bortezomib (BTZ) resistance in MM cells. Further study indicated overexpression of PFN1 significantly promoted the process of autophagy and induced BTZ resistance in MM. Otherwise, knockdown of PFN1 blocked autophagy and sensitized MM to BTZ. Co‐immunoprecipitation in MM cells indicated that PFN1 could bind Beclin1 complex and promote the initiation of autophagy. Inhibition of autophagy by blocking the formation of Beclin1 complex could reverse the phenotype of BTZ resistance in MM. Our findings suggested that PFN1 could promote autophagy through taking part in Beclin1 complex and contribute to BTZ resistance, which may become a novel molecular target in the therapy of MM.

A Positive Feedback Loop of Profilin-1 and RhoA/ROCK1 Promotes Endothelial Dysfunction and Oxidative Stress.

Vascular endothelial dysfunction is considered critical development in the progression of cardiovascular events and is associated with vascular damage and oxidative stress. Previous studies have shown that profilin-1 could be induced by advanced glycation end products (AGEs) and contributes to vascular hyperpermeability; however, the mechanisms are not fully understood. In this study, we sought to assess whether reactive oxygen species (ROS) were involved in profilin-1-mediated RhoA/ROCK1 signaling. Treatment with AGEs significantly induced the expression of profilin-1 and ROS production in human umbilical vein endothelial cells (HUVECs), whereas knockdown of profilin-1 diminished AGE-induced RhoA and ROCK1 activation and ROS production. Moreover, ectopic overexpression of profilin-1 also increased RhoA and ROCK1 activation and ROS production under low AGE concentration. Furthermore, blockage of RhoA/ROCK1 with the inhibitors CT04 and Y27632 significantly attenuated the profilin-1-mediated cell damage and ROS production. Additionally, ROS inhibition resulted in a significant decrease in profilin-1-mediated RhoA/ROCK1 expression, suggesting that the regulation of RhoA/ROCK1 signaling was partly independent of ROS. Taken together, these results suggested that the RhoA/ROCK1 pathway activated by excessive ROS is responsible for profilin-1-mediated endothelial damage.

ENOS S-nitrosylates β-actin on Cys374 and regulates PKC-θ at the immune synapse by impairing actin binding to profilin-1.

The actin cytoskeleton coordinates the organization of signaling microclusters at the immune synapse (IS); however, the mechanisms involved remain poorly understood. We show here that nitric oxide (NO) generated by endothelial nitric oxide synthase (eNOS) controls the coalescence of protein kinase C-θ (PKC-θ) at the central supramolecular activation cluster (c-SMAC) of the IS. eNOS translocated with the Golgi to the IS and partially colocalized with F-actin around the c-SMAC. This resulted in reduced actin polymerization and centripetal retrograde flow of β-actin and PKC-θ from the lamellipodium-like distal (d)-SMAC, promoting PKC-θ activation. Furthermore, eNOS-derived NO S-nitrosylated β-actin on Cys374 and impaired actin binding to profilin-1 (PFN1), as confirmed with the transnitrosylating agent S-nitroso-L-cysteine (Cys-NO). The importance of NO and the formation of PFN1-actin complexes on the regulation of PKC-θ was corroborated by overexpression of PFN1- and actin-binding defective mutants of β-actin (C374S) and PFN1 (H119E), respectively, which reduced the coalescence of PKC-θ at the c-SMAC. These findings unveil a novel NO-dependent mechanism by which the actin cytoskeleton controls the organization and activation of signaling microclusters at the IS.

Profilin-1 과발현에 의한 난소암 세포 성장 및 이동 저해 효능 연구

Abnormal actin remodeling is a typical characteristic of tumor cells. Thymosin β10 (TB10) and profilin-1 (PFN-1) are actin-binding proteins and essential regulators of actin polymerization. We previously showed that TB10 induced death in ovarian cancer cells by sequestering F-actin, but the underlying mechanisms of this induction have not been explored. In this study, we identified TB10 as a novel regulator of PFN-1 and demonstrated its novel function as a tumor suppressor in ovarian cancer cell lines. The present study investigated protein expression profiles through polyacrylamide gel electrophoresis (PAGE) and liquid chromatography-mass spectroscopy (LC-MS/MS) in SKOV3 cells, an ovarian cancer cell line, that were transiently transfected with TB10. PFN-1 was highly overexpressed in response to TB10, and overexpression of PFN-1 resulted in inhibition of cell proliferation and migration and promotion of cellular apoptosis in ovarian cancer cells. Furthermore, transiently transfected PFN-1 appeared to deactivate the Erk signaling pathway, followed by decreased expression of Elk-1 and Egr-1 in human ovarian cancer cells. Interestingly, PFN-1 did not affect the activation of Akt. The results demonstrated that PFN-1 induced apoptotic cell death and inhibited proliferation and migration in ovarian cancer cells, suggesting that PFN-1 may be valuable in anti-cancer therapy.

Profilin modulates sarcomeric organization and mediates cardiomyocyte hypertrophy.

AimsHeart failure is often preceded by cardiac hypertrophy, which is characterized by increased cell size, altered protein abundance, and actin cytoskeletal reorganization. Profilin is a well-conserved, ubiquitously expressed, multifunctional actin-binding protein, and its role in cardiomyocytes is largely unknown. Given its involvement in vascular hypertrophy, we aimed to test the hypothesis that profilin-1 is a key mediator of cardiomyocyte-specific hypertrophic remodelling.Methods and resultsProfilin-1 was elevated in multiple mouse models of hypertrophy, and a cardiomyocyte-specific increase of profilin in Drosophila resulted in significantly larger heart tube dimensions. Moreover, adenovirus-mediated overexpression of profilin-1 in neonatal rat ventricular myocytes (NRVMs) induced a hypertrophic response, measured by increased myocyte size and gene expression. Profilin-1 silencing suppressed the response in NRVMs stimulated with phenylephrine or endothelin-1. Mechanistically, we found that profilin-1 regulates hypertrophy, in part, through activation of the ERK1/2 signalling cascade. Confocal microscopy showed that profilin localized to the Z-line of Drosophila myofibrils under normal conditions and accumulated near the M-line when overexpressed. Elevated profilin levels resulted in elongated sarcomeres, myofibrillar disorganization, and sarcomeric disarray, which correlated with impaired muscle function.ConclusionOur results identify novel roles for profilin as an important mediator of cardiomyocyte hypertrophy. We show that overexpression of profilin is sufficient to induce cardiomyocyte hypertrophy and sarcomeric remodelling, and silencing of profilin attenuates the hypertrophic response.

Profilin 2 promotes migration, invasion, and stemness of HT29 human colorectal cancer stem cells.

We investigated the role of profilin 2 in the stemness, migration, and invasion of HT29 cancer stem cells (CSCs). Increased and decreased levels of profilin 2 significantly enhanced and suppressed the self-renewal, migration, and invasion ability of HT29 CSCs, respectively. Moreover, profilin 2 directly regulated the expression of stemness markers (CD133, SOX2, and β-catenin) and epithelial mesenchymal transition (EMT) markers (E-cadherin and snail). CD133 and β-catenin were up-regulated by overexpression of profilin 2 and down-regulated by depletion of profilin 2. SOX2 was decreased by profilin 2 depletion. E-cadherin was not influenced by profilin 2- overexpression but increased by profilin 2- knockdown. The expression of snail was suppressed by profilin 2- knockdown. We speculated that stemness and the EMT are closely linked through profilin 2-related pathways. Therefore, this study indicates that profilin 2 affects the metastatic potential and stemness of colorectal CSCs by regulating EMT- and stemness-related proteins.

Nicotine elevates sperm motility and induces Pfn1 promoter hypomethylation in mouse testis.

Many studies have addressed the hazardous role of cigarette smoking on male fertility, but the exact molecular mechanisms involved in the impairments caused by nicotine remain unclear. To evaluate the detrimental effects of nicotine exposure on spermatogenesis, two-dimensional gel electrophoresis and mass spectrometry analysis were performed to screen and identify differentially expressed proteins from the testes of mice exposed to nicotine daily. Data mining analysis indicated that the 15 identified proteins were mainly involved in actin cytoskeleton regulation and in the tricarboxylic acid cycle, which are related to cell motility. Further investigation of a central regulatory factor in the cytoskeleton regulation, profilin 1 (PFN1), revealed that nicotine-induced Pfn1 over-expression in mouse testes, specifically in elongated spermatids, by Pfn1 promoter hypomethylation. Interestingly, elevated sperm motility parameters were observed in nicotine-treated mice. We assume that nicotine-induced PFN1 over-expression in mouse spermatids may promote actin polymerization and ultimately enhance sperm motility.

Profilin-1 overexpression in MDA-MB-231 breast cancer cells is associated with alterations in proteomics biomarkers of cell proliferation, survival, and motility as revealed by global proteomics analyses.

Despite early screening programs and new therapeutic strategies, metastatic breast cancer is still the leading cause of cancer death in women in industrialized countries and regions. There is a need for novel biomarkers of susceptibility, progression, and therapeutic response. Global analyses or systems science approaches with omics technologies offer concrete ways forward in biomarker discovery for breast cancer. Previous studies have shown that expression of profilin-1 (PFN1), a ubiquitously expressed actin-binding protein, is downregulated in invasive and metastatic breast cancer. It has also been reported that PFN1 overexpression can suppress tumorigenic ability and motility/invasiveness of breast cancer cells. To obtain insights into the underlying molecular mechanisms of how elevating PFN1 level induces these phenotypic changes in breast cancer cells, we investigated the alteration in global protein expression profiles of breast cancer cells upon stable overexpression of PFN1 by a combination of three different proteome analysis methods (2-DE, iTRAQ, label-free). Using MDA-MB-231 as a model breast cancer cell line, we provide evidence that PFN1 overexpression is associated with alterations in the expression of proteins that have been functionally linked to cell proliferation (FKPB1A, HDGF, MIF, PRDX1, TXNRD1, LGALS1, STMN1, LASP1, S100A11, S100A6), survival (HSPE1, HSPB1, HSPD1, HSPA5 and PPIA, YWHAZ, CFL1, NME1) and motility (CFL1, CORO1B, PFN2, PLS3, FLNA, FLNB, NME2, ARHGDIB). In view of the pleotropic effects of PFN1 overexpression in breast cancer cells as suggested by these new findings, we propose that PFN1-induced phenotypic changes in cancer cells involve multiple mechanisms. Our data reported here might also offer innovative strategies for identification and validation of novel therapeutic targets and companion diagnostics for persons with, or susceptibility to, breast cancer.

Abstract 1325: Profilin-1 overexpression downregulates phosphorylation of p27kip1 leading to its nuclear accumulation in breast cancer cells

Abstract Profilin-1 is a ubiquitously expressed actin-binding protein that is downregulated in human breast cancer. Overexpression of profilin-1 in human breast cancer cells suppresses their tumorigenic potential. We had previously shown that profilin-1 overexpression elevates the expression level of p27kip1 leading to cell-cycle arrest in G1 phase. In the present work, we explore the underlying mechanism. Our studies showed that profilin-1-induced accumulation of p27kip1 in breast cancer cells occurs most dramatically in the nuclear compartment and this is due to impaired protein degradation. Consistent with these observations, we found that phosphorylation of p27kip1 at T187 (a prerequisite for p27kip1 degradation by SCF-Skp2 complex in the nucleus) and S10 (a post-translational event that promotes nuclear export of p27kip1) residues are both significantly inhibited by profilin-1 overexpression. Elevating profilin-1 level suppresses AKT activation, and hyper-activating AKT through overexpression of a constitutively active mutant relieves profilin-1-induced inhibition of T187 phosphorylation and elevation of p27kip1. These data demonstrate that profilin-1 can control the cellular level of p27kip1 in breast cancer cells through affecting its post-translational modification in an AKT-dependent pathway. These findings may provide mechanistic insights underlying the tumor-suppressive action of profilin-1. Citation Format: Chang Jiang, William Veon, Partha Roy. Profilin-1 overexpression downregulates phosphorylation of p27kip1 leading to its nuclear accumulation in breast cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1325. doi:10.1158/1538-7445.AM2014-1325

Profilin-1 Promotes the Development of Hypertension-induced Artery Remodeling

Hypertension is associated with the structural remodeling and stiffening of arteries and is known to increase cardiovascular risk. In the present study, we investigated the effects of overexpression and knock down of profilin-1 on the vascular structural remodeling in spontaneous hypertensive rats (SHRs) using an adenovirus injection to knock down or overexpress profilin-1 mRNA. As a control, blank adenovirus was injected into age-matched SHRs and Wistar-Kyoto rats (WKYs). We quantified arterial structural remodeling through morphological methods, with thoracic aortas stained with hematoxylin-eosin and picosirius red. Western blotting was performed to measure the protein expression of inducible nitric oxide synthase (iNOS) and p38 mitogen-activated protein kinase (p38), and peroxynitrite was quantified by immunohistochemical staining. Overexpression of profilin-1 significantly promoted aortic remodeling, including an increase in vessel size, wall thickness, and collagen content, whereas the knockdown of profilin-1 could reverse these effects. In addition, the expression of phosphorylated p38, iNOS and peroxynitrite was significantly upregulated in SHRs with profilin-1 overexpression along with an increased level of interleukin- 6 (IL-6). These changes could be reversed by knockdown of profilin-1. Our results demonstrate a crucial role for profilin-1 in hypertension-induced arterial structural remodeling at least in part through the p38-iNOS-peroxynitrite pathway.

A Legionella effector modulates host cytoskeletal structure by inhibiting actin polymerization

Successful infection by the opportunistic pathogen Legionella pneumophila requires the collective activity of hundreds of virulence proteins delivered into the host cell by the Dot/Icm type IV secretion system. These virulence proteins, also called effectors modulate distinct host cellular processes to create a membrane-bound niche called the Legionella containing vacuole (LCV) supportive of bacterial growth. We found that Ceg14 (Lpg0437), a Dot/Icm substrate is toxic to yeast and such toxicity can be alleviated by overexpression of profilin, a protein involved in cytoskeletal structure in eukaryotes. We further showed that mutations in profilin affect actin binding but not other functions such as interactions with poly-l-proline or phosphatidylinositol, abolish its suppressor activity. Consistent with the fact the profilin suppresses its toxicity, expression of Ceg14 but not its non-toxic mutants in yeast affects actin distribution and budding of daughter cells. Although Ceg14 does not detectably interact with profilin, it co-sediments with filamentous actin and inhibits actin polymerization, causing the accumulation of short actin filaments. Together with earlier studies, these results reveal that multiple L. pneumophila effectors target components of the host cytoskeleton.

Proteomic identification of Profilin1 as a corepressor of estrogen receptor alpha in MCF7 breast cancer cells

Nuclear receptor coregulators play an important role in the transcriptional regulation of nuclear receptors. In the present study, we aimed to identify estrogen receptor α (ERα) interacting proteins in Tamoxifen treated MCF7 cells. Using in vitro GST-pull down assay with ERα ligand-binding domain (ERα-LBD) and MS-based proteomics approach we identified Profilin1 as a novel ERα interacting protein. Profilin1 contains I/LXX/L/H/I amino acid signature motif required for corepressor interaction with ERα. We show that these two proteins physically interact with each other both in vitro as well as in vivo by GST-pull down and coimmunoprecipitation, respectively. We further show that these two proteins also colocalize together in the nucleus. Previous studies have reported reduced expression of Profilin1 in breast cancer; and here we found that Tamoxifen increases Profilin1 expression in MCF7 cells. Our data demonstrate that over expression of Profilin1 inhibits ERα-mediated transcriptional activation as well as its downstream target genes in ERα positive breast cancer cells MCF7. In addition, Profilin1 overexpression in MCF7 cells leads to inhibition of cell proliferation that apparently is due to enhanced apoptosis. In nutshell, these data indicate that MS-based proteomics approach identifies a novel ERα interacting protein Profilin1 that serves as a putative corepressor of ERα functions.