Profilin-1 Is Expressed in Human Atherosclerotic Plaques and Induces Atherogenic Effects on Vascular Smooth Muscle Cells

.Here we monitored profilin-1 expression in human atherosclerotic plaques by immunofluorescent staining. The effects of recombinant profilin-1 on atherogenic signaling pathways and cellular responses such as DNA synthesis (BrdU-incorporation) and chemotaxis (modified Boyden-chamber) were evaluated in cultured rat aortic and human coronary vascular smooth muscle cells (VSMCs). Furthermore, the correlation between profilin-1 serum levels and the degree of atherosclerosis was assessed in humans.<0.001 vs. no atherosclerosis or control group).Profilin-1 expression is significantly enhanced in human atherosclerotic plaques compared to the normal vessel wall, and the serum levels of profilin-1 correlate with the degree of atherosclerosis in humans. The atherogenic effects exerted by profilin-1 on VSMCs suggest an auto-/paracrine role within the plaque. These data indicate that profilin-1 might critically contribute to atherogenesis and may represent a novel therapeutic target

Suppression of Tumorigenicity in Breast Cancer Cells by the Microfilament Protein Profilin 1

Differential display screening was used to reveal differential gene expression between the tumorigenic breast cancer cell line CAL51 and nontumorigenic microcell hybrids obtained after transfer of human chromosome 17 into CAL51. The human profilin 1 (PFN1) gene was found overexpressed in the microcell hybrid clones compared with the parental line, which displayed a low profilin 1 level. A comparison between several different tumorigenic breast cancer cell lines with nontumorigenic lines showed consistently lower profilin 1 levels in the tumor cells. Transfection of PFN1 cDNA into CAL51 cells raised the profilin 1 level, had a prominent effect on cell growth, cytoskeletal organization and spreading, and suppressed tumorigenicity of the stable, PFN1-overexpressing cell clones in nude mice. Immunohistochemical analysis revealed intermediate and low levels of profilin 1 in different human breast cancers. These results suggest profilin 1 as a suppressor of the tumorigenic phenotype of breast cancer cells

Cancer-associated exportin-6 upregulation inhibits the transcriptionally repressive and anticancer effects of nuclear profilin-1

Aberrant expression of nuclear transporters and deregulated subcellular localization of their cargo proteins are emerging as drivers and therapeutic targets of cancer. Here, we present evidence that the nuclear exporter exportin-6 and its cargo profilin-1 constitute a functionally important and frequently deregulated axis in cancer. Exportin-6 upregulation occurs in numerous cancer types and is associated with poor patient survival. Reducing exportin-6 level in breast cancer cells triggers antitumor effects by accumulating nuclear profilin-1. Mechanistically, nuclear profilin-1 interacts with eleven-nineteen-leukemia protein (ENL) within the super elongation complex (SEC) and inhibits the ability of the SEC to drive transcription of numerous pro-cancer genes including MYC. XPO6 and MYC are positively correlated across diverse cancer types including breast cancer. Therapeutically, exportin-6 loss sensitizes breast cancer cells to the bromodomain and extra-terminal (BET) inhibitor JQ1. Thus, exportin-6 upregulation is a previously unrecognized cancer driver event by spatially inhibiting nuclear profilin-1 as a tumor suppressor

Mechanism of Profilin—1 in regulating eNOS/NO signaling pathway and its role in hypertensive myocardial hypertension

AbstractObjectiveTo explore the mechanism of Profilin-1 in regulating eNOS/NO pathway and its role in the development of myocardial hypertrophy.MethodsSpontaneously hypertensive rats (SHR) aged 5 weeks were injected with different adenovirus vectors to induce Profilin-1 expression knockdown (SHR-I) or over express (SHR-H) or to use as control (SHR-C). All these treatment were compared with Wistar-Kyoto rats (SKY) treated with control adenovirus vectors (WKY-C). The same injection was executed at the sixth week during the experiment of 12 weeks. After experiment, the left ventricular weight-to-heart weight ratio (LVW/HW) and left ventricular long axis (LVLA) were measured. Meanwhile, NO contents in blood and myocardium, Profilin-1, eNOS and Caveolin-3 mRNA and protein levels and phosphorylated eNOS (P-eNOS) protein level in myocardium were determined.ResultsCompared with WKY-C group, the SHR-C group was statistically higher in LVW/HW (0.79±0.03), LVLA (11.82±0.58 mm) and Profilin-1 mRNA and protein level (P<0.05), but lower in NO content [(18.63±6.23) μmol/L] in blood and [(2.71±0.17) μmol/L] in myocardium), eNOS activity and Caveolin-3 expression (P<0.05). The over expressing Profilin-1 led SHR-H group to a higher value of LVW/HW [(0.93±0.03) mm and LVLA (14.17±0.69) mm] in comparison with SHR-C group (P<0.05), and to a lower value of NO content (in myocardium), eNOS activity and Caveolin-3 expression (P<0.05); however, this phenomenon was reversed by the knockdown Profilin-1 expression (SHR-I group).ConclusionsProfilin-1 expression, being negative in regulating Caveolin-3 expression and eNOS/NO pathway activity, promotes the development of myocardial hypertrophy which can be reversed by Profilin-1 silencing

Profilin modulates sarcomeric organization and mediates cardiomyocyte hypertrophy

Aims: Heart 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, multi-functional actin-binding protein, whose 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 remodeling. Methods and Results: Profilin-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 signaling 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. Conclusion: Our 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 remodeling, and silencing of profilin attenuates the hypertrophic response

The Folding process of Human Profilin-1, a novel protein associated with familial amyotrophic lateral sclerosis

Human profilin-1 is a novel protein associated with a recently discovered form of familial amyotrophic lateral sclerosis. This urges the characterization of possible conformational states, different from the fully folded state, potentially able to initiate self-assembly. Under native conditions, profilin-1 is monomeric and possesses a well-defined secondary and tertiary structure. When incubated at low pH or with high urea concentrations, profilin-1 remains monomeric but populates unfolded states exhibiting larger hydrodynamic radius and disordered structure, as assessed by dynamic light scattering, far-UV circular dichroism and intrinsic fluorescence. Refolding from the urea-unfolded state was studied at equilibrium and in real-time using a stopped-flow apparatus. The results obtained with intrinsic fluorescence and circular dichroism indicate a single phase without significant changes of the corresponding signals before the major refolding transition. However, such a transition is preceded by a burst phase with an observed increase of ANS fluorescence, which indicates the conversion into a transiently populated collapsed state possessing solvent-exposed hydrophobic clusters. Kinetic analysis reveals that such state has a conformational stability comparable to that of the fully unfolded state. To our knowledge, profilin-1 is the first example of an amyloid-related protein where folding occurs in the absence of thermodynamically stable partially folded states

Quantitative image mean squared displacement (iMSD) analysis of the dynamics of profilin 1 at the membrane of live cells.

Image mean square displacement analysis (iMSD) is a method allowing the mapping of diffusion dynamics of molecules in living cells. However, it can also be used to obtain quantitative information on the diffusion processes of fluorescently labelled molecules and how their diffusion dynamics change when the cell environment is modified. In this paper, we describe the use of iMSD to obtain quantitative data of the diffusion dynamics of a small cytoskeletal protein, profilin 1 (pfn1), at the membrane of live cells and how its diffusion is perturbed when the cells are treated with Cytochalasin D and/or the interactions of pfn1 are modified when its actin and polyphosphoinositide binding sites are mutated (pfn1-R88A). Using total internal reflection fluorescence microscopy images, we obtained data on isotropic and confined diffusion coefficients, the proportion of cell areas where isotropic diffusion is the major diffusion mode compared to the confined diffusion mode, the size of the confinement zones and the size of the domains of dynamic partitioning of pfn1. Using these quantitative data, we could demonstrate a decreased isotropic diffusion coefficient for the cells treated with Cytochalasin D and for the pfn1-R88A mutant. We could also see changes in the modes of diffusion between the different conditions and changes in the size of the zones of pfn1 confinements for the pfn1 treated with Cytochalasin D. All of this information was acquired in only a few minutes of imaging per cell and without the need to record thousands of single molecule trajectories

SELDI-TOF-MS ProteinChip array profiling of T-cell clones propagated in long-term culture identifies human profilin-1 as a potential bio-marker of immunosenescence

<p>Abstract</p> <p>Background</p> <p>The adaptive immune response requires waves of T-cell clonal expansion on contact with pathogen and elimination after clearance of the source of antigen. However, lifelong persistent infections with common viruses cause chronic antigenic stimulation which takes its toll on adaptive immunity in late life. Chronic antigenic stress results in deregulation of the T-cell response and accumulation of anergic cells. Longitudinal studies of the elderly show that this impacts on survival. Identifying the nature of the defects in chronically-stimulated T-cells and protein bio-markers of these dysfunctional cells would help to understand age-associated compromised T-cell function (immunosenescence) and facilitate the development of targeted intervention strategies.</p> <p>The purpose of this work was to use surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) to analyse proteins associated with T-cell senescence in order to identify potential bio-markers. Clonal populations of T-cells isolated from elderly octogenarian and centenarian donors were grown <it>in vitro </it>until senescence, and early passage and late passage (pre-senescent) cells were analysed using SELDI-TOF-MS ProteinChip arrays.</p> <p>Results</p> <p>Discriminant analysis identified several protein or peptide peaks in the region of 14.5–16.5 kDa that were associated with T-cell clone senescence. Human profilin-1, a ubiquitous protein associated with actin remodelling and cellular motility was unambiguously identified. Altered expression of profilin-1 in senescent T-cell clones was confirmed by Western blot analysis.</p> <p>Conclusion</p> <p>Due to the proposed roles of profilin-1 in cellular survival, cytoskeleton remodelling, motility, and proliferation, it is hypothesised that differential expression of profilin-1 in ageing may contribute directly to immunosenescence.</p

ACE2 Deficiency Enhances Angiotensin II-Mediated Aortic Profilin-1 Expression, Inflammation and Peroxynitrite Production

Inflammation and oxidative stress play a crucial role in angiotensin (Ang) II-mediated vascular injury. Angiotensin-converting enzyme 2 (ACE2) has recently been identified as a specific Ang II-degrading enzyme but its role in vascular biology remains elusive. We hypothesized that loss of ACE2 would facilitate Ang II-mediated vascular inflammation and peroxynitrite production. 10-week wildtype (WT, Ace2+/y) and ACE2 knockout (ACE2KO, Ace2−/y) mice received with mini-osmotic pumps with Ang II (1.5 mg.kg−1.d−1) or saline for 2 weeks. Aortic ACE2 protein was obviously reduced in WT mice in response to Ang II related to increases in profilin-1 protein and plasma levels of Ang II and Ang-(1–7). Loss of ACE2 resulted in greater increases in Ang II-induced mRNA expressions of inflammatory cytokines monocyte chemoattractant protein-1 (MCP-1), interleukin (IL)-1β, and IL-6 without affecting tumor necrosis factor-α in aortas of ACE2KO mice. Furthermore, ACE2 deficiency led to greater increases in Ang II-mediated profilin-1 expression, NADPH oxidase activity, and superoxide and peroxynitrite production in the aortas of ACE2KO mice associated with enhanced phosphorylated levels of Akt, p70S6 kinase, extracellular signal-regulated kinases (ERK1/2) and endothelial nitric oxide synthase (eNOS). Interestingly, daily treatment with AT1 receptor blocker irbesartan (50 mg/kg) significantly prevented Ang II-mediated aortic profilin-1 expression, inflammation, and peroxynitrite production in WT mice with enhanced ACE2 levels and the suppression of the Akt-ERK-eNOS signaling pathways. Our findings reveal that ACE2 deficiency worsens Ang II-mediated aortic inflammation and peroxynitrite production associated with the augmentation of profilin-1 expression and the activation of the Akt-ERK-eNOS signaling, suggesting potential therapeutic approaches by enhancing ACE2 action for patients with vascular diseases