Effect of profilin-1 on the asymmetric dimethylarginine-induced vascular lesion-associated hypertension.

Previous studies have demonstrated that the levels of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide (NO) synthesis, are strongly associated with hypertension, diabetes, and cardiovascular diseases. Profilin-1, an actin-binding protein, has been documented to be involved in endothelial injury and in the proliferation of vascular smooth muscle cells resulting from hypertension. However, the role of profilin-1 in ADMA-induced vascular injury in hypertension remains largely unknown. Forty healthy subjects and forty-two matched patients with essential hypertension were enrolled, and the related indexes of vascular injury in plasma were detected. Rat aortic smooth muscle cells (RASMCs) were treated with different concentrations of ADMA for different periods of time and transfected with profilin-1 small hairpin RNA to interrupt the expression of profilin-1. To determine the role of the Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) pathway, RASMCs were pretreated with AG490 or rapamycin. The expression of profilin-1 was tested using real-time polymerase chain reaction (PCR) and western blot analysis. Cell proliferation was measured by flow cytometry and 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazoliumbromide assays. Compared with healthy subjects, the levels of ADMA and profilin-1 were markedly elevated in hypertensive individuals, while the levels of NO were significantly decreased (p < 0.05). In vitro, studies showed ADMA-induced profilin-1 expression in a concentration- and time-dependent manner in RASMCs (p < 0.05), concomitantly with promoting the proliferation of RASMCs. Furthermore, ADMA-mediated proliferation of RASMCs and upregulation expression of profilin-1 were inhibited by blockade of the JAK2/STAT3 pathway or knockdown of profilin-1. Profilin-1 implicated in the ADMA-mediated vascular lesions in hypertension.

Coordinated activity of amoebic formin and profilin are essential for phagocytosis.

For the protist parasite Entamoeba histolytica, endocytic processes, such as phagocytosis, are essential for its survival in the human gut. The actin cytoskeleton is involved in the formation of pseudopods and phagosomal vesicles by incorporating a number of actin-binding and modulating proteins along with actin in a temporal manner. The actin dynamics, which comprises polymerization, branching, and depolymerization is very tightly regulated and takes place directionally at the sites of initiation of phagocytosis. Formin and profilin are two actin-binding proteins that are known to regulate actin cytoskeleton dynamics and thereby, endocytic processes. In this article, we report the participation of formin and profilin in E. histolytica phagocytosis and propose that these two proteins interact with each other and their sequential recruitment at the site is required for the successful completion of phagocytosis. The evidence is based on detailed microscopic, live imaging, interaction studies, and expression downregulation. The cells downregulated for expression of formin show absence of profilin at the site of phagocytosis, whereas downregulation of profilin does not affect formin localization.

Arp2/3 and Mena/VASP Require Profilin 1 for Actin Network Assembly at the Leading Edge.

Cells have many types of actin structures, which must assemble from a common monomer pool. Yet, it remains poorly understood how monomers are distributed to and shared between different filament networks. Simplified model systems suggest that monomers are limited and heterogeneous, which alters actin network assembly through biased polymerization and internetwork competition. However, less is known about how monomers influence complex actin structures, where different networks competing for monomers overlap and are functionally interdependent. One example is the leading edge of migrating cells, which contains filament networks generated by multiple assembly factors. The leading edge dynamically switches between the formation of different actin structures, such as lamellipodia or filopodia, by altering the balance of these assembly factors' activities. Here, we sought to determine how the monomer-binding protein profilin 1 (PFN1) controls the assembly and organization of actin in mammalian cells. Actin polymerization in PFN1 knockout cells was severely disrupted, particularly at the leading edge, where both Arp2/3 and Mena/VASP-based filament assembly was inhibited. Further studies showed that in the absence of PFN1, Arp2/3 no longer localizes to the leading edge and Mena/VASP is non-functional. Additionally, we discovered that discrete stages of internetwork competition and collaboration between Arp2/3 and Mena/VASP networks exist at different PFN1 concentrations. Low levels of PFN1 caused filopodia to form exclusively at the leading edge, while higher concentrations inhibited filopodia and favored lamellipodia and pre-filopodia bundles. These results demonstrate that dramatic changes to actin architecture can be made simply by modifying PFN1 availability.

Human antigen R-regulated mRNA metabolism promotes the cell motility of migrating mouse neurons.

Neocortex development during embryonic stages requires the precise control of mRNA metabolism. Human antigen R (HuR) is a well-studied mRNA-binding protein that regulates mRNA metabolism, and it is highly expressed in the neocortex during developmental stages. Deletion of HuR does not impair neural progenitor cell proliferation or differentiation, but it disturbs the laminar structure of the neocortex. We report that HuR is expressed in postmitotic projection neurons during mouse brain development. Specifically, depletion of HuR in these neurons led to a mislocalization of CDP+ neurons in deeper layers of the cortex. Time-lapse microscopy showed that HuR was required for the promotion of cell motility in migrating neurons. PCR array identified profilin 1 (Pfn1) mRNA as a major binding partner of HuR in neurons. HuR positively mediated the stability of Pfn1 mRNA and influenced actin polymerization. Overexpression of Pfn1 successfully rescued the migration defects of HuR-deleted neurons. Our data reveal a post-transcriptional mechanism that maintains actin dynamics during neuronal migration.

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.

Profilin Directly Promotes Microtubule Growth through Residues Mutated in Amyotrophic Lateral Sclerosis.

Profilin is an abundant actin monomer-binding protein with critical actin regulatory roles in vivo [1, 2]. However, profilin also influences microtubule dynamics in cells, which may be mediated in part through its interactions with formins that in turn bind microtubules [3, 4]. Specific residues on human profilin-1 (PFN1) are mutated in patients with amyotrophic lateral sclerosis (ALS) [5, 6]. However, the observation that some ALS-linked PFN1 mutants fail to alter cellular actin organization or dynamics [5-8] or in vitro actin-monomer affinity [9] has been perplexing, given that profilin is best understood as an actin regulator. Here, we investigated direct effects of profilin on microtubule dynamics and whether ALS-linked mutations in PFN1 disrupt such functions. We found that human, fly, and yeast profilin homologs all directly enhance microtubule growth rate by several-fold in vitro. Microtubule stimulatory effects were unaffected by mutations in the canonical actin- or poly-proline-binding sites of profilin. Instead, microtubule activities depended on specific surface residues on profilin mutated in ALS patients. Furthermore, microtubule effects were attenuated by increasing concentrations of actin monomers, suggesting competition between actin and microtubules for binding profilin. Consistent with these biochemical observations, a 2-fold increase in the expression level of wild-type PFN1, but not the ALS-linked PFN1 mutants, increased microtubule growth rates in cells. Together, these results demonstrate that profilin directly enhances the growth rate of microtubules. They further suggest that ALS-linked mutations in PFN1 may perturb cellular microtubule dynamics and/or the coordination between the actin and microtubule cytoskeletons, leading to motor neuron degeneration.

Intrinsic, Functional, and Structural Properties of ß-Thymosins and ß-Thymosin/WH2 Domains in the Regulation and Coordination of Actin Self-Assembly Dynamics and Cytoskeleton Remodeling.

ß-Thymosins are a family of heat-stable multifunctional polypeptides that are expressed as small proteins of about 5kDa (~45 amino acids) almost exclusively in multicellular animals. They were first isolated from the thymus. As full-length or truncated polypeptides, they appear to stimulate a broad range of extracellular activities in various signaling pathways, including tissue repair and regeneration, inflammation, cell migration, and immune defense. However, their cell surface receptors and structural mechanisms of regulations in these multiple pathways remain still poorly understood. Besides their extracellular activities, they belong to a larger family of small, intrinsically disordered actin-binding domains called WH2/ß-thymosin domains that have been identified in more than 1800 multidomain proteins found in different taxonomic domains of life and involved in various actin-based motile processes including cell morphogenesis, motility, adhesions, tissue development, intracellular trafficking, or pathogen infections. This review briefly surveys the main recent findings to understand how these small, intrinsically disordered but functional domains can interact with many unrelated partners and can thus integrate and coordinate various intracellular activities in actin self-assembly dynamics and cell signaling pathways linked to their cytoskeleton remodeling.

Actoclampin (+)-end-tracking motors: How the pursuit of profilin's role(s) in actin-based motility twice led to the discovery of how cells crawl.

The path to the discovery of the actoclampins began with efforts to define profilin's role in actin-based pathogen and endosome rocketing. That research identified a set of FPPPP-containing cargo proteins and FPPPP-binding proteins that are consistently stationed within the polymerization zone during episodes of active motility. The very same biophysical clues that forced us to abandon Brownian Ratchet models guided us to the Actoclampin Hypothesis, which asserts that every propulsive filament possesses a (+)-end-tracking motor that generates the forces cells need to crawl. Each actoclampin motor is a multi-arm oligomeric complex, employing one arm to recruit/deliver Profilin•Actin•ATP to a growth-site located at the (+)-end of the lagging subfilament, while a second arm maintains an affinity-modulated binding interaction with the extreme (+)-end of the other subfilament. The alternating actions of these arms define a true molecular motor, the processivity of which explains why propelling filaments maintain full possession of their cargo. The Actoclampin Hypothesis also suggests how the energetics of tracker interactions with the (+)-end determines whether a given actoclampin is a passive (low force-producing) or active (high force-producing) motor, the latter requiring the Gibbs free energy of ATP hydrolysis. Another aim of this review is to acknowledge an earlier notional model that emerged from efforts to comprehend profilin's pivotal role(s) in actin-based cell motility.

Regulation of contractile ring formation and septation in Schizosaccharomyces pombe.

The fission yeast Schizosaccharomyces pombe has become a powerful model organism for cytokinesis studies, propelled by pioneering genetic screens in the 1980s and 1990s. S. pombe cells are rod-shaped and divide similarly to mammalian cells, utilizing a medially-placed actin-and myosin-based contractile ring. A cell wall division septum is deposited behind the constricting ring, forming the new ends of each daughter cell. Here we discuss recent advances in our understanding of the regulation of contractile ring formation through formin proteins and the role of the division septum in S. pombe cell division.

Epithelial morphological reversion drives Profilin-1-induced elevation of p27(kip1) in mesenchymal triple-negative human breast cancer cells through AMP-activated protein kinase activation.

Profilin-1 (Pfn1) is an important regulator of actin polymerization that is downregulated in human breast cancer. Previous studies have shown Pfn1 has a tumor-suppressive effect on mesenchymal-like triple-negative breast cancer cells, and Pfn1-induced growth suppression is partly mediated by upregulation of cell-cycle inhibitor p27(kip1) (p27). In this study, we demonstrate that Pfn1 overexpression leads to accumulation of p27 through promoting AMPK activation and AMPK-dependent phosphorylation of p27 on T198 residue, a post-translational modification that leads to increased protein stabilization of p27. This pathway is mediated by Pfn1-induced epithelial morphological reversion of mesenchymal breast cancer through cadherin-mediated restoration of adherens junctions. These findings not only elucidate a potential mechanism of how Pfn1 may inhibit proliferation of mesenchymal breast cancer cells, but also highlight a novel pathway of cadherin-mediated p27 induction and therefore cell-cycle control in cells.

RhoA/mDia-1/profilin-1 signaling targets microvascular endothelial dysfunction in diabetic retinopathy.

BACKGROUND: Diabetic retinopathy (DR) is a major cause of blindness in the working-age populations of developed countries, and effective treatments and prevention measures have long been the foci of study. Patients with DR invariably demonstrate impairments of the retinal microvascular endothelium. Many observational and preclinical studies have shown that angiogenesis and apoptosis play crucial roles in the pathogenesis of DR. Increasing evidence suggests that in DR, the small guanosine-5'-triphosphate-binding protein RhoA activates its downstream targets mammalian Diaphanous homolog 1 (mDia-1) and profilin-1, thus affecting important cellular functions, including cell morphology, motility, secretion, proliferation, and gene expression. However, the specific underlying mechanism of disease remains unclear. CONCLUSION: This review focuses on the RhoA/mDia-1/profilin-1 signaling pathway that specifically triggers endothelial dysfunction in diabetic patients. Recently, RhoA and profilin-1 signaling has attracted a great deal of attention in the context of diabetes-related research. However, the precise molecular mechanism by which the RhoA/mDia-1/profilin-1 pathway is involved in progression of microvascular endothelial dysfunction (MVED) during DR has not been determined. This review briefly describes each feature of the cascade before exploring the most recent findings on how the pathway may trigger endothelial dysfunction in DR. When the underlying mechanisms are understood, novel therapies seeking to restore the endothelial homeostasis comprised in DR will become possible.

Profilin 1 is required for peripheral nervous system myelination.

Myelination allows rapid saltatory propagation of action potentials along the axon and is an essential prerequisite for the normal functioning of the nervous system. During peripheral nervous system (PNS) development, myelin-forming Schwann cells (SCs) generate radial lamellipodia to sort and ensheath axons. This process requires controlled cytoskeletal remodeling, and we show that SC lamellipodia formation depends on the function of profilin 1 (Pfn1), an actin-binding protein involved in microfilament polymerization. Pfn1 is inhibited upon phosphorylation by ROCK, a downstream effector of the integrin linked kinase pathway. Thus, a dramatic reduction of radial lamellipodia formation is observed in SCs lacking integrin-linked kinase or treated with the Rho/ROCK activator lysophosphatidic acid. Knocking down Pfn1 expression by lentiviral-mediated shRNA delivery impairs SC lamellipodia formation in vitro, suggesting a direct role for this protein in PNS myelination. Indeed, SC-specific gene ablation of Pfn1 in mice led to profound radial sorting and myelination defects, confirming a central role for this protein in PNS development. Our data identify Pfn1 as a key effector of the integrin linked kinase/Rho/ROCK pathway. This pathway, acting in parallel with integrin ß1/LCK/Rac1 and their effectors critically regulates SC lamellipodia formation, radial sorting and myelination during peripheral nervous system maturation.

Profilin 1 is essential for retention and metabolism of mouse hematopoietic stem cells in bone marrow.

How stem cells interact with the microenvironment to regulate their cell fates and metabolism is largely unknown. Here we demonstrated that the deletion of the cytoskeleton-modulating protein profilin 1 (pfn1) in hematopoietic stem cell (HSCs) led to bone marrow failure, loss of quiescence, and mobilization and apoptosis of HSCs in vivo. A switch from glycolysis to mitochondrial respiration with increased reactive oxygen species (ROS) level was also observed in HSCs on pfn1 deletion. Importantly, treatment of pfn1-deficient mice with the antioxidant N-acetyl-l-cysteine reversed the ROS level and loss of quiescence of HSCs, suggesting that the metabolism is mechanistically linked to the cell cycle quiescence of stem cells. The actin-binding and proline-binding activities of pfn1 are required for its function in HSCs. Our study provided evidence that pfn1 at least partially acts through the axis of pfn1/Gα13/EGR1 to regulate stem cell retention and metabolism in the bone marrow.

Interaction of profilin with the barbed end of actin filaments.

Profilin binds not only to actin monomers but also to the barbed end of the actin filament, where it inhibits association of subunits. To address open questions about the interactions of profilin with barbed ends, we measured the effects of a wide range of concentrations of Homo sapiens profilin 1 on the rate of elongation of individual skeletal muscle actin filaments by total internal reflection fluorescence microscopy. Much higher concentrations of profilin were required to stop elongation by AMP-PNP-actin monomers than ADP-actin monomers. High concentrations of profilin depolymerized barbed ends at a rate much faster than the spontaneous dissociation rates of Mg-ATP-, Mg-AMP-PNP-, Mg-ADP-Pi-, and Mg-ADP-actin subunits. Fitting a thermodynamic model to these data allowed us to determine the affinities of profilin and profilin-actin for barbed ends and the influence of the nucleotide bound to actin on these interactions. Profilin has a much higher affinity for ADP-actin filament barbed ends (Kd = 1 µM) than AMP-PNP-actin filament barbed ends (Kd = 226 µM). ADP-actin monomers associated with profilin bind to ADP-actin filament barbed ends 10% as fast as free ADP-actin monomers, but bound profilin does not affect the rate of association of AMP-PNP-actin monomers with barbed ends. The differences in the affinities of AMP-PNP- and ADP-bound barbed ends for profilin and profilin-actin suggest that conformations of barbed end subunits differ from those of monomers and change upon nucleotide hydrolysis and phosphate release. A structural model revealed minor steric clashes between profilin and actin subunits at the barbed end that explain the biochemical results.

Zm908p11, encoded by a short open reading frame (sORF) gene, functions in pollen tube growth as a profilin ligand in maize.

Double fertilization of flowering plants depends on the targeted transportation of sperm to the embryo sac by the pollen tube. Currently, little is known about the underlying molecular mechanisms that regulate pollen germination and pollen tube growth in maize (Zea mays). Here, a maize pollen-predominant gene Zm908, with several putative short open reading frames (sORFs), was isolated and characterized. The longest ORF of Zm908 encodes a small protein of 97 amino acids. This was designated as Zm908p11 and is distributed throughout the maize pollen tube. Western blot detected the small peptide in mature pollen. Quantitative reverse transcription-PCR and northern blot analysis revealed that Zm908p11 was expressed predominantly in mature pollen grains. Ectopic overexpression of full-length Zm908 and Zm908p11 in tobacco resulted in defective pollen, while transgenic tobacco plants with a site-specific mutation or a frameshift mutation of Zm908p11 showed normal pollen development. Overexpression of Zm908p11 in maize decreased pollen germination efficiency. Maize pollen cDNA library screening and protein-protein interaction assays demonstrated that Zm908p11 interacts with maize profilin 1 (ZmPRO1). A microarray analysis identified 273 up-regulated and 203 down-regulated genes in the overexpressing transgenic Zm908p11 pollen. Taken together, these results indicate that Zm908 functions as Zm908p11, and binds to profilins as a novel ligand, with a required role during pollen tube growth in maize. Accordingly, a model is proposed for the role of Zm908p11 during pollen tube growth in maize.

Profilin-1 promotes the development of hypertension-induced cardiac hypertrophy.

OBJECTIVE: Cardiac hypertrophy is a major cause of heart failure and sudden cardiac death among hypertensive individuals. The present study examined the effects of profilin-1 on hypertension-induced cardiac hypertrophy. METHODS: We used adenovirus injection to knockdown or overexpress profilin-1 in spontaneous hypertensive rats (SHRs). As a control, blank adenovirus was injected into age-matched SHRs and Wistar-Kyoto rats (WKYs). SBP and cardiac mass index were measured. Cardiac tissues were stained with hematoxylin-eosin and sirius red, and cardiac ultrastructure was imaged using transmission electron microscopy. Actin filament was quantified by staining with TRIC-tagged phalloidin. Caveolin-3 abundance and endothelial nitric oxide synthase (eNOS) activity were measured using real-time quantitative PCR, Western blot or immunofluorescence staining. RESULTS: Endogenous profilin-1 was highly expressed in hypertrophic myocardium of SHRs compared with WKYs. Lowering profilin-1 expression in SHRs significantly attenuated hypertension-induced cardiac hypertrophy and fibrosis and displayed a significant preservation of myofibrils, sarcolemmal caveolae, abundance of caveolin-3 protein, activity of eNOS and production of nitric oxide (NO). In contrast, transgenic overexpression of profilin-1 in SHRs induced more serious cardiac hypertrophy and fibrosis with significant reduction of sarcolemmal caveolae, caveolin-3 protein, eNOS activity, and production of NO when compared with SHR controls. CONCLUSION: Profilin-1 promotes cardiac hypertrophy partly through interfering with the formation of sarcolemmal caveolae and attenuating the eNOS/NO pathway. These results demonstrate a crucial role for profilin-1 in hypertensive cardiac hypertrophy.

Differential remodeling of actin cytoskeleton architecture by profilin isoforms leads to distinct effects on cell migration and invasion.

Dynamic actin cytoskeletal reorganization is integral to cell motility. Profilins are well-characterized regulators of actin polymerization; however, functional differences among coexpressed profilin isoforms are not well defined. Here, we demonstrate that profilin-1 and profilin-2 differentially regulate membrane protrusion, motility, and invasion; these processes are promoted by profilin-1 and suppressed by profilin-2. Compared to profilin-1, profilin-2 preferentially drives actin polymerization by the Ena/VASP protein, EVL. Profilin-2 and EVL suppress protrusive activity and cell motility by an actomyosin contractility-dependent mechanism. Importantly, EVL or profilin-2 downregulation enhances invasion in vitro and in vivo. In human breast cancer, lower EVL expression correlates with high invasiveness and poor patient outcome. We propose that profilin-2/EVL-mediated actin polymerization enhances actin bundling and suppresses breast cancer cell invasion.

Profilin1 regulates sternum development and endochondral bone formation.

Bone development is a dynamic process that requires cell motility and morphological adaptation under the control of actin cytoskeleton. This actin cytoskeleton system is regulated by critical modulators including actin-binding proteins. Among them, profilin1 (Pfn1) is a key player to control actin fiber structure, and it is involved in a number of cellular activities such as migration. During the early phase of body development, skeletal stem cells and osteoblastic progenitor cells migrate to form initial rudiments for future skeletons. During this migration, these cells extend their process based on actin cytoskeletal rearrangement to locate themselves in an appropriate location within microenvironment. However, the role of Pfn1 in regulation of mesenchymal progenitor cells (MPCs) during skeletal development is incompletely understood. Here we examined the role of Pfn1 in skeletal development using a genetic ablation of Pfn1 in MPCs by using Prx1-Cre recombinase. We found that Pfn1 deficiency in MPCs caused complete cleft sternum. Notably, Pfn1-deficient mice exhibited an absence of trabecular bone in the marrow space of appendicular long bone. This phenotype is location-specific, as Pfn1 deficiency did not largely affect osteoblasts in cortical bone. Pfn1 deficiency also suppressed longitudinal growth of long bone. In vitro, Pfn1 deficiency induced retardation of osteoblastic cell migration. These observations revealed that Pfn1 is a critical molecule for the skeletal development, and this could be at least in part associated with the retardation of cell migration.

Actin filament attachments for sustained motility in vitro are maintained by filament bundling.

We reconstructed cellular motility in vitro from individual proteins to investigate how actin filaments are organized at the leading edge. Using total internal reflection fluorescence microscopy of actin filaments, we tested how profilin, Arp2/3, and capping protein (CP) function together to propel thin glass nanofibers or beads coated with N-WASP WCA domains. Thin nanofibers produced wide comet tails that showed more structural variation in actin filament organization than did bead substrates. During sustained motility, physiological concentrations of Mg(2+) generated actin filament bundles that processively attached to the nanofiber. Reduction of total Mg(2+) abolished particle motility and actin attachment to the particle surface without affecting actin polymerization, Arp2/3 nucleation, or filament capping. Analysis of similar motility of microspheres showed that loss of filament bundling did not affect actin shell formation or symmetry breaking but eliminated sustained attachments between the comet tail and the particle surface. Addition of Mg(2+), Lys-Lys(2+), or fascin restored both comet tail attachment and sustained particle motility in low Mg(2+) buffers. TIRF microscopic analysis of filaments captured by WCA-coated beads in the absence of Arp2/3, profilin, and CP showed that filament bundling by polycation or fascin addition increased barbed end capture by WCA domains. We propose a model in which CP directs barbed ends toward the leading edge and polycation-induced filament bundling sustains processive barbed end attachment to the leading edge.