Profilin Expression Research Articles

Phleum pratense-pollen adaptive variations and pollen microbiome investigation under different climatic regions and prospects of allergenicity.

Phleum pratense is an allergenic grass that pollinates in spring in Pakistan. Databases Allergenonline.org and Allergen.org record ten P. pratense allergens and their isoforms. Phl P 1, Phlp 5, and Phl p 11 are major P. pratense-pollen allergens with demonstrated basophil activity and skin test reactivity. Little is known about P. pratense pollen adaptive variations in different climatic regions and pollen-associated microbial diversity. In this study, we collected P. pratense-pollen and soils in the spring season 2022. Samples were collected from three climatic regions in Pakistan (R1, R2 and R3) with differences in mean monthly air temperature, mean monthly precipitation and elevation. The morphology of pollen was observed by light microscopy, scanning electron microscopy (SEM), biochemical fingerprint analysis, and composition of pollen were investigated by fourier-transform infrared spectroscopy (FTIR). The pollen-associated bacterial populations were identified through a Biolog GEN III microplate system. The pollen water-soluble proteins were isolated and stabilized in phosphate buffer saline (PBS) and tested for allergenicity responses through dot blots and western blots analysis. The morphology study found difference in pollen biochemical composition. Biolog identified Brevibacterium epidermidis and Pantoea agglomerans from P. pratense pollen. Protein extract quantification and sodium dodecyl sulfate-poly acrylamide gel electrophoresis (SDS-PAGE) gel found decreased protein expression in R1 region pollen compared to R2 and R3 region pollen. Allergenicity studies found differential expression of beta-expansin and profilin allergens in pollen obtained from the three regions. Beta-expansin and profilin were suppressed in R1 pollen and expressed in compared to R2 and R3 pollen. This is the first study to identify B. epidermidis and P. agglomerans growth on P. pratense pollen. Variable allergen expression in P. pratense pollen has also been observed in different regions. Soil pH, an increase in mean monthly temperature and a decrease in mean monthly precipitation correlated with pollen biochemical composition, and reduced beta-expansin and profilin expression involved in pollen growth and development. The findings of this research are unique, which enhances basic knowledge and understanding of P. pratense-pollen associated microbiota and climate change impacts on the pollen allergen expression.

Profilin upregulation induces autophagy through stabilization of AMP-activated protein kinase.

Profilin regulates actin polymerization, and its balanced expression is required for cellular growth and development. Most tumours have compromised profilin expression, and its overexpression in MDA MB-231 breast cancer cells has been reported to activate AMP-activated protein kinase α (AMPKα), an energy-sensing molecule that affects various cellular processes including autophagy. This study aims to explore the role of profilin in autophagy induction. We employed all-trans retinoic acid (ATRA) as an inducer of profilin expression and showed that profilin induces autophagy through mTOR inhibition, autophagy-activating kinase ULK1 upregulation, and AMPK stabilization as well as its activation. Furthermore, evidence from our study indicates physical interaction between profilin and AMPK, which results in AMPK stabilization and induction of prolonged autophagy, thereby leading to apoptosis. This study uncovers a novel mechanism that induces autophagy in triple-negative breast cancer cells.

Increased expression of Profilin potentiates chemotherapeutic agent-mediated tumour regression.

Targeted cancer therapy is an alternative to standard chemotherapy for a better prognosis. Although its incompetency for triple-negative breast cancer (TNBC), treatment still relies on classical chemotherapy. Increasing evidence suggest that chemotherapeutic drug-induced toxic effect could be minimised by combinatorial therapy. Profilin's familiar anti-tumorigenic activity can be utilised in combination with the drug to improve efficacy, which could be promising therapeutics to treat TNBC. All-trans retinoic acid (ATRA) in combination with vinblastine was tested on human MDA MB-231 cell line (MB-231) (in vitro) and MB-231 borne breast cancer in nude mice (in vivo). Effects of combination treatment on tumour growth inhibition and apoptosis were examined by tumour volume, histology and PARP cleavage. ATRA-induced transcriptional regulation of profilin had been evaluated by gel-shift and reporter gene assays. Profilin's role in ATRA-induced vinblastine efficacy was validated in profilin-stable and profilin-silenced cells. ATRA binds with RAR/RXR to increase the profilin expression that potentiated cell death by chemotherapeutics. ATRA priming led to vinblastine-mediated potentiation of G2-M phase cell cycle arrest in MB-231 cells and regression of breast cancer in xenograft mice at very low concentration without any adverse effects. Moreover, increased p53 and PTEN but downregulated p65 in the tumour tissues further supported the involvement of profilin for tumour regression. Vinblastine at very low concentration (20 times lesser than the recommended dose for breast cancer therapeutic) significantly regress tumour growth in ATRA-primed mice without any toxic effects suggesting potential combinatorial therapeutics for TNBC.

Relationship between the Levels of mRNA Expression for Protein Phosphatase 1B and Proteins Involved in Cytoskeleton Remodeling in Squamous Cell Carcinoma of the Larynx and Hypopharynx.

We analyzed the expression of genes encoding proteins involved in cytoskeleton remodeling (RND3, SNAI1, vimentin, cofilin, adenylate cyclase-associated protein 1, ezrin, and profilin) depending on the level of expression of protein phosphatase 1B (PPM1B) mRNA on the example of squamous cell carcinoma of the larynx and hypopharynx. Against the background of a high level of PPM1B expression, a significantly high level of profilin expression was noted. Metastasis correlated with the level of snai1 expression, while relapse after combination treatment was negatively associated with the level of vimentin expression. The obtained new data can reflect molecular peculiarities of the tumor growth in squamous cell carcinoma of the larynx and hypopharynx.

Profilin: many facets of a small protein

Profilin is a ubiquitously expressed protein well known as a key regulator of actin polymerisation. The actin cytoskeleton is involved in almost all cellular processes including motility, endocytosis, metabolism, signal transduction and gene transcription. Hence, profilin's role in the cell goes beyond its direct and essential function in regulating actin dynamics. This review will focus on the interactions of Profilin 1 and its ligands at the plasma membrane, in the cytoplasm and the nucleus of the cells and the regulation of profilin activity within those cell compartments. We will discuss the interactions of profilin in cell signalling pathways and highlight the importance of the cell context in the multiple functions that this small essential protein has in conjunction with its role in cytoskeletal organisation and dynamics. We will review some of the mechanisms that control profilin expression and the implications of changed expression of profilin in the light of cancer biology and other pathologies.

Synchronous Transcription of Cytoskeleton-Associated Genes is Critical to Cotton Fiber Elongation

Cytoskeleton-associated genes’ expression in the modern cotton fibers has selectively been evolved under domestication. Since the magnitude of transcriptional dynamicity of cytoskeletal genes in fibers has not yet been thoroughly explored, comprehensive expression analyses of the domestication-driven profilins (GhPRFs) were performed by assaying 12 tissues from various developmental stages of elite cotton genotypes. Results from 3240 successful profilin (GhPRF1–GhPRF6) expression assays revealed enormous spatial transcriptional biases with their predominant expression in the elongating fibers. Moreover, fiber developmental stages have more pronounced impacts on differential gene expression than the genetic backgrounds. Transcriptional comparisons of GhPRFs-associated key cytoskeletal genes GhAnnex and GhACT show perfect (r = 1.0) to strong positive (r > 0.95) correlation in the developing fibers. RNAi-mediated constitutive suppression of domestication-driven GhPRF1 gene resulted in significant expression modulation of GhAnnex and GhACT genes in floral/fiber tissues and consequently sustained the cell wall development. These results highlight that temporal expression of GhPRF–GhAnnex–GhACT triad is exceptionally coordinated which illustrates fascinating crosstalk in diverse cellular contexts and cell types. Temporal fold-expression variations of other cytoskeletal-‘structural’ genes Tub–Exp–FLA triad were transversely synchronous to the GhPRF–GhAnnex–GhACT genic triad. However, the CMW-‘modifying’ genes GalT1, ABP1, and CesA were unconditionally divergent from the synchronous temporal dynamics of genic triad investigated beforehand. To our knowledge, this study represents the first in-depth description of synchronous transcriptional dynamicity of cytoskeletal-‘structural’ genes in the developing fibers. These results present a novel viewpoint on the temporal expression evolution of cytoskeletal-‘structural’ genes, augmenting our understanding of the evolutionary genetics of cytoskeletal genes in fiber development.

TLR11 or TLR12 silencing reduces Leishmania major infection

Toll-like receptors (TLRs) recognize the pathogen-associated molecular patterns (PAMPs) and induce host-protective immune response. The role of the profilin-recognizing TLR11/TLR12 in Leishmania infection is unknown. Herein, we report that TLR11/ TLR12 expression increases in virulent L. major-infected macrophages but is prevented by miltefosine, an anti-leishmanial drug. While lipohosphoglycan (LPG) increases, LPG or TLR2 blockade prevents, the heightened TLR11/TLR12 expression. LPG-TLR2 interaction triggers MyD88- and TIRAP-mediated signaling enhancing ERK-1/2 activation and increased production of IL-10 that promotes TLR11/TLR12 expression. Profilin expression was higher in the virulent L. major and L. donovani parasites than that observed in the avirulent parasites. TLR11 or TLR12 silencing reduces parasite burden and increases IFN-γ, but reduces IL-4, production indicating that TLR11 and TLR12 play a pro-leishmanial role.

Profilin connects actin assembly with microtubule dynamics.

Profilin controls actin nucleation and assembly processes in eukaryotic cells. Actin nucleation and elongation promoting factors (NEPFs) such as Ena/VASP, formins, and WASP-family proteins recruit profilin:actin for filament formation. Some of these are found to be microtubule associated, making actin polymerization from microtubule-associated platforms possible. Microtubules are implicated in focal adhesion turnover, cell polarity establishment, and migration, illustrating the coupling between actin and microtubule systems. Here we demonstrate that profilin is functionally linked to microtubules with formins and point to formins as major mediators of this association. To reach this conclusion, we combined different fluorescence microscopy techniques, including superresolution microscopy, with siRNA modulation of profilin expression and drug treatments to interfere with actin dynamics. Our studies show that profilin dynamically associates with microtubules and this fraction of profilin contributes to balance actin assembly during homeostatic cell growth and affects micro-tubule dynamics. Hence profilin functions as a regulator of microtubule (+)-end turnover in addition to being an actin control element.

Profilin Expression Is Regulated by Bone Morphogenetic Protein (BMP) in Osteoblastic Cells.

Profilin 1 (Pfn1) regulates cytoskeletal reorganization and migration, but its role in osteoblasts is not known. BMP (bone morphogenetic protein) is a multifunctional cytokine involved in osteoblastic differentiation and promotes bone regeneration and repair. Although several molecules are known to modulate BMP signaling, mechanisms that determine the levels of BMP action in osteoblastic function are still incompletely understood. We therefore examine the expression of Pfn1 in osteoblasts and its role in BMP-induced differentiation in osteoblasts. In osteoblastic MC3T3-E1(MC) cells, Pfn1 mRNA is expressed constitutively and its expression levels are declined during the culture in a time dependent manner in contrast to the increase in alkaline phosphatase activity revealing that Pfn1 expression is down regulated along with differentiation. To test the effects of osteoblastic differentiation on Pfn1expression further, MC cells are treated with BMP. BMP treatment suppresses the levels of Pfn1 mRNA. This suppressive effect of BMP is time dependent and further down regulation of Pfn1 mRNA levels is observed when the BMP treatment is continued for a longer period of time. Pfn1mRNA knock down (KD) by siRNAs enhances BMP-induced increase in alkaline phosphatase (Alp) activity in MC cells. To analyze the regulatory mechanism, Alp mRNA levels are examined and Pfn1 KD enhances the BMP-induced increase in the levels of Alp mRNA expression. Furthermore, Pfn1 KD enhances BMP-induced transcriptional expression of luciferase reporter activity via BMP response element in osteoblasts. These data indicate that Pfn1 is a novel target of BMP and suppresses BMP-induced differentiation of osteoblasts at least in part via transcriptional event.

Urokinase-type plasminogen activator promotes dendritic spine recovery and improves neurological outcome following ischemic stroke.

Spines are dendritic protrusions that receive most of the excitatory input in the brain. Early after the onset of cerebral ischemia dendritic spines in the peri-infarct cortex are replaced by areas of focal swelling, and their re-emergence from these varicosities is associated with neurological recovery after acute ischemic stroke (AIS). Urokinase-type plasminogen activator (uPA) is a serine proteinase that plays a central role in tissue remodeling via binding to the urokinase plasminogen activator receptor (uPAR). We report that cerebral cortical neurons release uPA during the recovery phase from ischemic stroke in vivo or hypoxia in vitro. Although uPA does not have an effect on ischemia- or hypoxia-induced neuronal death, genetic deficiency of uPA (uPA(-/-)) or uPAR (uPAR(-/-)) abrogates functional recovery after AIS. Treatment with recombinant uPA after ischemic stroke induces neurological recovery in wild-type and uPA(-/-) but not in uPAR(-/-) mice. Diffusion tensor imaging studies indicate that uPA(-/-) mice have increased water diffusivity and decreased anisotropy associated with impaired dendritic spine recovery and decreased length of distal neurites in the peri-infarct cortex. We found that the excitotoxic injury induces the clustering of uPAR in dendritic varicosities, and that the binding of uPA to uPAR promotes the reorganization of the actin cytoskeleton and re-emergence of dendritic filopodia from uPAR-enriched varicosities. This effect is independent of uPA's proteolytic properties and instead is mediated by Rac-regulated profilin expression and cofilin phosphorylation. Our data indicate that binding of uPA to uPAR promotes dendritic spine recovery and improves functional outcome following AIS.

Variability of Corylus Avellana, L. CorA and profilin pollen allergens expression

Corylus avellana is the source of inhalant allergies induced by hazel pollen as well as food allergies induced after ingestion of hazelnuts. In this study, real-time PCR approach was used to analyse expression of hazel pollen allergens on the molecular level. Relative quantity of hazelnut allergens Corylus avellana, L. CorA and Corylus avellana, L. pollen profiling in samples from different Ukraine areas were determining and comparing. Differences among the levels of both analysed allergen transcripts were found for hazel CorA and profillin. In both cases, the expression within the urbanized growth conditions was higher when compared to the sample from village area. The average expression for CorA was 0.84 times higher than for profilin and the results are very variable depending on the place of growth. Expression levels here were within the range of 2.957 up to the 52.936. Profilin expression was the highest in the sample from the polluted place of growth—cement plant area with the value of 52 times higher when compared to the sample from the village area. In this study, comparison of expression levels of hazel CorA and profiling pollen allergens was performed for the first time. Real-time PCR assay developed in this study proved the sensitivity for detection of the changes of the hazel pollen allergens expression levels and could benefit labs by fast and reproducible detection method of these allergens.

Parallel up-regulation of the profilin gene family following independent domestication of diploid and allopolyploid cotton ( Gossypium )

Cotton is remarkable among our major crops in that four species were independently domesticated, two allopolyploids and two diploids. In each case thousands of years of human selection transformed sparsely flowering, perennial shrubs into highly productive crops with seeds bearing the vastly elongated and abundant single-celled hairs that comprise modern cotton fiber. The genetic underpinnings of these transformations are largely unknown, but comparative gene expression profiling experiments have demonstrated up-regulation of profilin accompanying domestication in all three species for which wild forms are known. Profilins are actin monomer binding proteins that are important in cytoskeletal dynamics and in cotton fiber elongation. We show that Gossypium diploids contain six profilin genes (GPRF1-GPRF6), located on four different chromosomes (eight chromosomes in the allopolyploid). All but one profilin (GPRF6) are expressed during cotton fiber development, and both homeologs of GPRF1-GPRF5 are expressed in fibers of the allopolyploids. Remarkably, quantitative RT-PCR and RNAseq data demonstrate that GPRF1-GPRF5 are all up-regulated, in parallel, in the three independently domesticated cottons in comparison with their wild counterparts. This result was additionally supported by iTRAQ proteomic data. In the allopolyploids, there This usage of novel should be fine, since it refers to a novel evolutionary process, not a novel discovery has been novel recruitment of the sixth profilin gene (GPRF6) as a result of domestication. This parallel up-regulation of an entire gene family in multiple species in response to strong directional selection is without precedent and suggests unwitting selection on one or more upstream transcription factors or other proteins that coordinately exercise control over profilin expression.

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

BackgroundProfilin-1 is an ubiquitous actin binding protein. Under pathological conditions such as diabetes, profilin-1 levels are increased in the vascular endothelium. We recently demonstrated that profilin-1 overexpression triggers indicators of endothelial dysfunction downstream of LDL signaling, and that attenuated expression of profilin-1 confers protection from atherosclerosis in vivo.MethodologyHere 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.Principal FindingsIn coronary arteries from patients with coronary heart disease, we found markedly enhanced profilin expression in atherosclerotic plaques compared to the normal vessel wall. Stimulation of rat aortic and human coronary VSMCs with recombinant profilin-1 (10−6 M) in vitro led to activation of intracellular signaling cascades such as phosphorylation of Erk1/2, p70S6 kinase and PI3K/Akt within 10 minutes. Furthermore, profilin-1 concentration-dependently induced DNA-synthesis and migration of both rat and human VSMCs, respectively. Inhibition of PI3K (Wortmannin, LY294002) or Src-family kinases (SU6656, PP2), but not PLCγ (U73122), completely abolished profilin-induced cell cycle progression, whereas PI3K inhibition partially reduced the chemotactic response. Finally, we found that profilin-1 serum levels were significantly elevated in patients with severe atherosclerosis in humans (p<0.001 vs. no atherosclerosis or control group).ConclusionsProfilin-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.

Target validation and ligand development for a pathogenic fungal profilin, using a knock‐down strain of pathogenic yeast Candida glabrata and structure‐based ligand design

The emergence of antifungal drug resistance is triggering vigorous searches for novel antifungal targets and lead compounds. In this study, we focused on fungal profilin, which is a small actin control protein sharing limited homology to human profilin. To validate its potentiality as a target, a profilin-conditional mutant of the pathogenic yeast Candida glabrata was constructed, using a regulatable Tet promoter, and its growth was monitored in vitro. Repression of profilin expression led to severe growth defect, demonstrating the potential of this protein as a novel antifungal target. Next, novel peptides binding to the active interface of profilin were designed by computer simulation. ELISA analysis showed that these peptides did bind to the wild-type profilin but bound less strongly to a profilin with amino acid substitutions at the active interface. Hence, we show here that profilin is a potential antifungal target and offer novel peptide ligands.

Differential expression of microfilamental network-associated proteins in circulating leukocytes of patients with benign and malignant breast tumors versus controls as revealed by clinical proteomics: potential application to early and predictive diagnosis.

Abstract Abstract #2037 Background: Breast cancer is one of the most frequent tumors world-wide. Early diagnosis has been demonstrated to be highly beneficial for significantly enhanced therapeutic efficiency and possibly full recovery. Currently applied diagnostic approaches are frequently not able to recognize very early stages in tumor development that impair the outcome. Our efforts are focused on the creation of early and even predictive diagnosis of breast cancer based on an application of advanced minimal-invasive molecular biotechnological approaches such as pathology-specific blood proteome.&amp;#x2028; Material and methods: Protein expression profiling was performed in circulating leukocytes isolated from fresh blood samples of age-matched female patients with invasive breast cancer (9 invasive lobular and 10 invasive ductal tumors), benign breast tumors (11 patients) and 9 healthy controls. In patients with all kinds of breast tumors, blood samples were taken before core needle biopsy of the lump. Comparative protein mapping in circulating leukocytes was performed by 2D-PAGE followed by MALDI-TOF-analysis of differentially expressed protein spots. Corresponding expression levels of identified proteins were further measured using Western-blot quantification.&amp;#x2028; Results: Molecular patterns were identified in circulating leukocytes and found to be specific for breast tumor patients. The results achieved at all levels were highly reproducible. The pathology specific patterns comprise microfilamental network-associated proteins: Calgranulins, LyGDI, RhoA, profilin, and actins. Calgranulin, RhoA, and profilin expression levels were significantly higher in healthy controls compared to patients with breast tumors, regardless the tumor malignancy (p=0.002 p&amp;lt;0.001 and p=0.025, respectively). Moreover, calgranulin expression levels discriminated well among lobular carcinomas, benign tumors, ductal carcinomas, and controls. Strong correlation between profilin and RhoA expression levels was demonstrated in all individual samples. Noteworthy, significant difference for expression levels of actins between patients with both malignant and benign tumors versus controls was demonstrated.&amp;#x2028; Conclusions: Microfilamental network-associated proteins identified here are involved in the regulation of a variety of cellular processes; they are functionally linked with each other and known to be highly relevant for all stages of tumorigenesis beginning with precancerous lesions and including activated metastasing potential. Statistical analysis demonstrated high specificity of the tumor-related molecular patterns. These findings are currently considered for a development of novel highly sensitive minimal-invasive approaches for early diagnosis of breast cancer. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 2037.

Expression Analysis and Characteristics of Profilin Gene from Silkworm, Bombyx mori

A recombinant Bombyx mori profilin protein (rBmPFN) was overexpressed in Escherichia coli BL21. Purified rBmPFN was used to generate anti-BmPFN polyclonal antibody, which were used to determine the subcellular localization of BmPFN. Immunostaining indicated that profilin can be found in both the nucleus and cytoplasm but is primarily located in the cytoplasm. Real-time RT-PCR and Western blot analyses indicated that, during the larvae stage, profilin expression levels are highest in the silk gland, followed by the gonad, and are lowest in the fatty body. Additionally, BmPFN expression begins during the egg stage, increases during the larvae stage, reaches a peak during the pupa stage, and decreases significantly in the moth. Therefore, we propose that BmPFN may play an important role during larva stage development, especially in the silk gland.

Caenorhabditis elegans expresses three functional profilins in a tissue‐specific manner

Profilins are actin binding proteins, which also interact with polyphosphoinositides and proline-rich ligands. On the basis of the genome sequence, three diverse profilin homologues (PFN) are predicted to exist in Caenorhabditis elegans. We show that all three isoforms PFN-1, PFN-2, and PFN-3 are expressed in vivo and biochemical studies indicate they bind actin and influence actin dynamics in a similar manner. In addition, they bind poly(L-proline) and phosphatidylinositol 4,5-bisphosphate micelles. PFN-1 is essential whereas PFN-2 and PFN-3 are nonessential. Immunostainings revealed different expression patterns for the profilin isoforms. In embryos, PFN-1 localizes in the cytoplasm and to the cell-cell contacts at the early stages, and in the nerve ring during later stages. During late embryogenesis, expression of PFN-3 was specifically detected in body wall muscle cells. In adult worms, PFN-1 is expressed in the neurons, the vulva, and the somatic gonad, PFN-2 in the intestinal wall, the spermatheca, and the pharynx, and PFN-3 localizes in a striking dot-like fashion in body wall muscle. Thus the model organism Caenorhabditis elegans expresses three profilin isoforms and is the first invertebrate animal with tissue-specific profilin expression.

Profilin acts downstream of LDL to mediate diabetic endothelial cell dysfunction

The changes occurring at the luminal surface of endothelial cells in diabetes and their relevance to endothelial dysfunction are poorly characterized in vivo. In this study, we developed an integrated strategy to discover cell surface proteins associated with diabetes and to test their role in endothelial dysfunction. First, a peptide phage display library was screened over the endothelial surface of the intact aorta or in retinal endothelial cells from diabetic and control rats. Then, we purified profilin-1 as a binding partner for one of the diabetic aorta-specific phage. Profilin was increased in the aortic endothelium of human diabetic individuals and streptozotocin-diabetic rats. Furthermore, overexpressing profilin in rat aortic endothelial cells triggered 3 indicators of endothelial dysfunction: increased apoptosis, elevated expression of ICAM-1, and decreased phosphorylation of the vasodilator-stimulated phosphoprotein, a marker for nitric oxide signaling. The changes in ICAM-1 and vasodilator-stimulated phosphoprotein were recapitulated in the diabetic aorta in vivo. LDL and oxysterols elevated profilin in cultured aortic endothelial cells. Interference with the de novo synthesis of profilin abrogated the LDL-mediated increase in ICAM-1 expression. Finally, profilin expression was markedly elevated in atherosclerotic plaques. These data indicate that profilin contributes to endothelial dysfunction in a pathway that is downstream of LDL.

Expression of profilin, an actin-binding protein, in rat experimental glomerulonephritis and its upregulation by basic fibroblast growth factor in cultured rat mesangial cells.

Profilin binds to actin monomer to regulate actin polymerization, and to phosphatidylinositol 4,5-bisphosphate to inhibit hydrolysis by phospholipase Cgamma1. This study investigated the expression of profilin in rat anti-Thy-1.1 mesangial proliferative glomerulonephritis (GN) and examined the effect of growth factors on its expression in cultured rat mesangial cells. Profilin mRNA was constitutively expressed in isolated glomeruli of untreated rats. However, in glomeruli of anti-Thy-1.1 GN rats, its expression was upregulated beginning on day 1, reaching a peak level on day 4 (3.9-fold versus control glomeruli), and decreased on day 14, as determined by competitive reverse transcription-PCR. Increased expression of profilin protein was confirmed using immunoblotting and immunohistochemistry. Immunoelectron microscopy revealed the presence of profilin in plasma membrane and the rough endoplasmic reticulum of mesangial cells, indicating that profilin was produced in mesangial cells. In cultured rat mesangial cells, expression of profilin mRNA and protein was upregulated by basic fibroblast growth factor but not by platelet-derived growth factor or transforming growth factor-beta. Suppression of profilin expression using an antisense oligonucleotide against profilin inhibited [3H]thymidine uptake. These findings indicated the involvement of profilin in anti-Thy-1.1 GN and suggest that the upregulation of profilin might be involved in the progression of anti-Thy-1.1 GN possibly by affecting cell growth.

Coordinate Induction of the Actin Cytoskeletal Regulatory Proteins Gelsolin, Vasodilator-Stimulated Phosphoprotein, and Profilin during Capillary Morphogenesisin Vitro

The formation of capillaries during development and tissue repair is likely to involve active reorganization of the actin cytoskeleton, although few studies have addressed this issue. Here, we have utilized anin vitromodel of capillary morphogenesis whereby human umbilical vein endothelial cells are suspended within three-dimensional type I collagen gels. The cells undergo dramatic morphogenic changes to develop capillary lumens, tubes, and networks over 72 h of culture. Western blots using cell extracts of these gels over this time frame were performed using antibodies directed to various proteins associated with the actin cytoskeleton. Three proteins showed altered expression during the time course, and they were gelsolin, which increased fivefold; vasodilator-stimulated phosphoprotein (VASP), which increased twofold; and profilin, which increased threefold in expression between the 24- and the 72-h time points. Reverse transcriptase-polymerase chain reaction and Northern blot analysis revealed a similar increase in mRNA expression of the three proteins. After the onset of network formation, the differentiated endothelial cells (dECs) undergoing capillary morphogenesis were removed from collagen gels at 48 h of culture to compare their properties with untreated endothelial cells (uECs). These dECs showed two- to threefold increased spontaneous migration in Boyden chamber assays compared to uECs. The dECs also displayed a prominent spindle-shaped morphology and the novel presence of intranuclear gelsolin compared to uECs when both cell types were replated on type I collagen-coated microwells and glass coverslips. These data suggest that increased gelsolin, VASP, and profilin expression may play an important role in the regulation of capillary tube and network formation in three-dimensional extracellular matrix.