Biological evaluation of sulfonate and sulfate analogues of lithocholic acid: A bioisosterism-guided approach towards the discovery of potential sialyltransferase inhibitors for antimetastatic study.

The naturally occurring bile acid lithocholic acid (LCA) has been a crucial core structure for many non-sugar-containing sialyltranferase (ST) inhibitors documented in literature. With the aim of elucidating the impact of the terminal carboxyl acid substituent of LCA on its ST inhibition, in this present study, we report the (bio)isosteric replacement-based design and synthesis of sulfonate and sulfate analogues of LCA. Among these compounds, the sulfate analogue SPP-002 was found to selectively inhibit N-glycan sialylation by at least an order of magnitude, indicating a substantial improvement in both potency and selectivity when compared to the unmodified parent bile acid. Molecular docking analysis supported the stronger binding of the synthetic analogue in the enzyme active site. Treatment with SPP-002 also hampered the migration, adhesion, and invasion of MDA-MB-231 cells in vitro by suppressing the expression of signaling proteins involved in the cancer metastasis-associated integrin/FAK/paxillin pathway. In totality, these findings offer not only a novel structural scaffold but also valuable insights for the future development of more potent and selective ST inhibitors with potential therapeutic effects against tumor cancer metastasis.

Progerin Inhibits the Proliferation and Migration of Melanoma Cells by Regulating the Expression of Paxillin.

Objective: Progerin, the underlying cause of Hutchinson-Gilford Progeria Syndrome (HGPS), has been extensively studied for its impact on normal cells and premature aging patients. However, there is a lack of research on its specific effects on tumor cells. Melanoma is one of the most common malignant tumors with high morbidity and mortality. This study aimed to elucidate the potential therapeutic role of progerin in melanoma. Materials and Methods: We constructed the melanoma A375 cell line and M14 cell line with stable expression of progerin. The expression of progerin, paxillin, and epithelial-mesenchymal transition (EMT) marker proteins in each cell group was measured using Western blot. The migration, proliferation, and cell cycle of cancer cells were assessed using the transwell assay, wound healing assay, colony formation assay, CCK 8 assay, and flow cytometry. RT-qPCR technology was used to examine the impact of progerin overexpression on microRNA expression. Finally, we transfected paxillin into the progerin overexpression cell group to verify whether progerin regulates the phenotype of tumor cells through paxillin. Results: Our study demonstrated that overexpression of progerin leads to decreased expression of paxillin and inhibits cancer cell migration, proliferation, EMT process and cell cycle progression. Additionally, rescue experiments revealed that the migration, proliferation ability, and EMT marker protein expression in progerin overexpressing cancer cells could be partially restored by transfecting a plasmid containing the paxillin gene. Mechanistic investigations further revealed that progerin achieves this inhibition of paxillin expression by upregulating miR-212. Conclusion: This study reveals that progerin may inhibit the migration and proliferation of melanoma cells through the miR-212/paxillin axis, which provides a new approach for the future treatment of this disease.

Role of steroid hormones in the maintenance of focal adhesions in bovine oviductal epithelial cells.

The oviduct, the organ of the female reproductive system where fertilization and early embryonic development occur, provides an optimal environment for the final maturation of oocytes, storage, and sperm capacitation and transport of gametes and embryos. During the estrous cycle, the oviduct is affected by ovarian sex hormones, resulting in changes aimed at maintaining an appropriate microenvironment. Normal cell migration is tightly regulated, its role being essential for the development and maintenance of organ and tissue functions as well as for regeneration following injury. Due to their involvement in focal contact formations, focal adhesion kinase (PTK2) and paxillin (PXN) are key proteins in the study of cell migration and adhesion. The objective of this work was to compare the expression of PTK2 and PXN in oviductal cells along the estrous cycle and to determine if their expression is regulated by the presence of 17-ß estradiol (E2) and/or progesterone (P4). No transcripts of PTK2 or of PXN were detected in cells corresponding to the luteal phase. Additionally, hormonal stimulation experiments on bovine oviductal cell cultures (BOECs) were carried out, where P4 inhibited the expression of both genes. Migration assays demonstrated that P4 reduced BOECs migration capacity. P4 treatment also reduced cell adhesion, while E2 increased the number of adhered cells. In conclusion, the presence of E2 and P4 regulates the expression of genes involved in the formation of focal contacts and modifies the migration and adhesion of BOECs. Understanding the effect of steroid hormones on BOECs is critical to grasp the impact of steroid control on oviductal function and its contribution to establishing successful pregnancies.

ICAM-1 Deletion Using CRISPR/Cas9 Protects the Brain from Traumatic Brain Injury-Induced Inflammatory Leukocyte Adhesion and Transmigration Cascades by Attenuating the Paxillin/FAK-Dependent Rho GTPase Pathway.

Intercellular adhesion molecule-1 (ICAM-1) is identified as an initiator of neuroinflammatory responses that lead to neurodegeneration and cognitive and sensory-motor deficits in several pathophysiological conditions including traumatic brain injury (TBI). However, the underlying mechanisms of ICAM-1-mediated leukocyte adhesion and transmigration and its link with neuroinflammation and functional deficits following TBI remain elusive. Here, we hypothesize that blocking of ICAM-1 attenuates the transmigration of leukocytes to the brain and promotes functional recovery after TBI. The experimental TBI was induced in vivo by fluid percussion injury (25 psi) in male and female wild-type and ICAM-1-/- mice and in vitro by stretch injury (3 psi) in human brain microvascular endothelial cells (hBMVECs). We treated hBMVECs and animals with ICAM-1 CRISPR/Cas9 and conducted several biochemical analyses and demonstrated that CRISPR/Cas9-mediated ICAM-1 deletion mitigates blood-brain barrier (BBB) damage and leukocyte transmigration to the brain by attenuating the paxillin/focal adhesion kinase (FAK)-dependent Rho GTPase pathway. For analyzing functional outcomes, we used a cohort of behavioral tests that included sensorimotor functions, psychological stress analyses, and spatial memory and learning following TBI. In conclusion, this study could establish the significance of deletion or blocking of ICAM-1 in transforming into a novel preventive approach against the pathophysiology of TBI.

Effect of Paxillin Expression and Phosphorylation on Colorectal Cancer Prognosis and Metastasis.

BACKGROUND/AIM: Colorectal cancer (CRC) is the third most common cancer worldwide, and metastasis is strongly associated with poor prognosis in patients with CRC. We have previously found that the expression and phosphorylation of paxillin (PXN) play an important role in the metastatic potential of breast cancer. This study examined the potential role of PXN in CRC metastasis. MATERIALS AND METHODS: Resected tumor specimens from 92 patients with CRC were subjected to immunohistochemical analysis of PXN levels. Three human CRC cell lines, HCT116, LoVo, and SW480 were used for scratch and transwell invasion assays to examine the effects of PXN over-expression. RNA sequencing was performed to obtain the expression profiles under PXN over-expression. RESULTS: High levels of PXN were significantly correlated with advanced stage, higher carcinoembryonic antigen and carbohydrate antigen 19-9 levels, and poorer overall survival. The migration ability of CRC cells was enhanced by exogenous PXN over-expression, but this enhancement was not observed in cells harboring exogenously mutated PXN at Tyr31 or Tyr88 phosphorylation sites. In PXN-over-expressing cells, TNF-α signaling via NF-[Formula: see text]B was positively enriched. CONCLUSION: PXN expression and phosphorylation at Tyr31 or Tyr88 may influence the migration and invasion of CRC cells. PXN expression and phosphorylation at Tyr31 or Tyr88 are promising targets for evaluating prognosis and treating CRC.

Cell-Material Interplay in Focal Adhesion Points.

The complex interplay between cells and materials is a key focus of this research, aiming to develop optimal scaffolds for regenerative medicine. The need for tissue regeneration underscores understanding cellular behavior on scaffolds, especially cell adhesion to polymer fibers forming focal adhesions. Key proteins, paxillin and vinculin, regulate cell signaling, migration, and mechanotransduction in response to the extracellular environment. This study utilizes advanced microscopy, specifically the AiryScan technique, along with advanced image analysis employing the Density-Based Spatial Clustering of Applications with Noise (DBSCAN) cluster algorithm, to investigate protein distribution during osteoblast cell adhesion to polymer fibers and glass substrates. During cell attachment to both glass and polymer fibers, a noticeable shift in the local maxima of paxillin and vinculin signals is observed at the adhesion sites. The focal adhesion sites on polymer fibers are smaller and elliptical but exhibit higher protein density than on the typical glass surface. The characteristics of focal adhesions, influenced by paxillin and vinculin, such as size and density, can potentially reflect the strength and stability of cell adhesion. Efficient adhesion correlates with well-organized, larger focal adhesions characterized by increased accumulation of paxillin and vinculin. These findings offer promising implications for enhancing scaffold design, evaluating adhesion to various substrates, and refining cellular interactions in biomedical applications.

Role of the Ste20-like kinase SLK in podocyte adhesion.

SLK controls the cytoskeleton, cell adhesion, and migration. Podocyte-specific deletion of SLK in mice leads to podocyte injury as mice age and exacerbates injury in experimental focal segment glomerulosclerosis (FSGS; adriamycin nephrosis). We hypothesized that adhesion proteins may be substrates of SLK. In adriamycin nephrosis, podocyte ultrastructural injury was exaggerated by SLK deletion. Analysis of a protein kinase phosphorylation site dataset showed that podocyte adhesion proteins-paxillin, vinculin, and talin-1 may be potential SLK substrates. In cultured podocytes, deletion of SLK increased adhesion to collagen. Analysis of paxillin, vinculin, and talin-1 showed that SLK deletion reduced focal adhesion complexes (FACs) containing these proteins mainly in adriamycin-induced injury; there was no change in FAC turnover (focal adhesion kinase Y397 phosphorylation). In podocytes, paxillin S250 showed basal phosphorylation that was slightly enhanced by SLK; however, SLK did not phosphorylate talin-1. In adriamycin nephrosis, SLK deletion did not alter glomerular expression/localization of talin-1 and vinculin, but increased focal adhesion kinase phosphorylation modestly. Therefore, SLK decreases podocyte adhesion, but FAC proteins in podocytes are not major substrates of SLK in health and disease.

Paxillin participates in the sphingosylphosphorylcholine-induced abnormal contraction of vascular smooth muscle by regulating Rho-kinase activation.

BACKGROUND: The Ca2+-independent contraction of vascular smooth muscle is a leading cause of cardiovascular and cerebrovascular spasms. In the previous study, we demonstrated the involvement of Src family protein tyrosine kinase Fyn and Rho-kinase in the sphingosylphosphorylcholine (SPC)-induced abnormal and Ca2+-independent contraction of vascular smooth muscle, but the specific mechanism has not been completely clarified. METHODS: Paxillin knockdown human coronary artery smooth muscle cells (CASMCs) and smooth muscle-specific paxillin knockout mice were generated by using paxillin shRNA and the tamoxifen-inducible Cre-LoxP system, respectively. CASMCs contraction was observed by time-lapse recording. The vessel contractility was measured by using a myography assay. Fyn, Rho-kinase, and myosin light chain activation were assessed by immunoprecipitation and western blotting. The paxillin expression and actin stress fibers were visualized by histological analysis and immunofluorescent staining. RESULTS: The SPC-induced abnormal contraction was inhibited in paxillin knockdown CASMCs and arteries of paxillin knockout mice, indicating that paxillin is involved in this abnormal contraction. Further study showed that paxillin knockdown inhibited the SPC-induced Rho-kinase activation without affecting Fyn activation. In addition, paxillin knockdown significantly inhibited the SPC-induced actin stress fiber formation and myosin light chain phosphorylation. These results suggest that paxillin, as an upstream molecule of Rho-kinase, is involved in the SPC-induced abnormal contraction of vascular smooth muscle. CONCLUSIONS: The present study demonstrated that paxillin participates in the SPC-induced abnormal vascular smooth muscle contraction by regulating Rho-kinase activation. Video Abstract.

Hic-5 regulates extracellular matrix-associated gene expression and cytokine secretion in cancer associated fibroblasts.

The focal adhesion protein, Hic-5 plays a key role in promoting extracellular matrix deposition and remodeling by cancer associated fibroblasts within the tumor stroma to promote breast tumor cell invasion. However, whether stromal matrix gene expression is regulated by Hic-5 is still unknown. Utilizing a constitutive Hic-5 knockout, Mouse Mammary Tumor Virus-Polyoma Middle T-Antigen spontaneous breast tumor mouse model, bulk RNAseq analysis was performed on cancer associated fibroblasts isolated from Hic-5 knockout mammary tumors. Functional network analysis highlighted a key role for Hic-5 in extracellular matrix organization, with both structural matrix genes, as well as matrix remodeling genes being differentially expressed in relation to Hic-5 expression. The subcellular distribution of the MRTF-A transcription factor and expression of a subset of MRTF-A responsive genes was also impacted by Hic-5 expression. Additionally, cytokine array analysis of conditioned media from the Hic-5 and Hic-5 knockout cancer associated fibroblasts revealed that Hic-5 is important for the secretion of several key factors that are associated with matrix remodeling, angiogenesis and immune evasion. Together, these data provide further evidence of a central role for Hic-5 expression in cancer associated fibroblasts in regulating the composition and organization of the tumor stroma microenvironment to promote breast tumor progression.

Paxillin/HDAC6 regulates microtubule acetylation to promote directional migration of keratinocytes driven by electric fields.

Endogenous electric fields (EFs) have been demonstrated to facilitate wound healing by directing the migration of epidermal cells. Despite the identification of numerous molecules and signaling pathways that are crucial for the directional migration of keratinocytes under EFs, the underlying molecular mechanisms remain undefined. Previous studies have indicated that microtubule (MT) acetylation is linked to cell migration, while Paxillin exerts a significant influence on cell motility. Therefore, we postulated that Paxillin could enhance EF-induced directional migration of keratinocytes by modulating MT acetylation. In the present study, we observed that EFs (200 mV/mm) induced migration of human immortalized epidermal cells (HaCaT) towards the anode, while upregulating Paxillin, downregulating HDAC6, and increasing the level of microtubule acetylation. Our findings suggested that Paxillin plays a pivotal role in inhibiting HDAC6-mediated microtubule acetylation during directional migration under EF regulation. Conversely, downregulation of Paxillin decreased microtubule acetylation and electrotaxis of epidermal cells by promoting HDAC6 expression, and this effect could be reversed by the addition of tubacin, an HDAC6-specific inhibitor. Furthermore, we observed that EFs also mediated the polarization of Paxillin and acetylated α-tubulin, which is critical for directional migration. In conclusion, our study revealed that MT acetylation in EF-guided keratinocyte migration is regulated by the Paxillin/HDAC6 signaling pathway, providing a novel theoretical foundation for the molecular mechanism of EF-guided directional migration of keratinocytes.

Lidocaine inhibits migration of tenocytes by downregulating focal adhesion kinase and paxillin phosphorylation.

Lidocaine is the most frequently applied local infiltration anesthetic agent for treating tendinopathies. However, studies have discovered lidocaine to negatively affect tendon healing. In the current study, the molecular mechanisms and effects of lidocaine on tenocyte migration were evaluated. We treated tenocytes intrinsic to the Achilles tendons of Sprague-Dawley rats with lidocaine. The migration ability of cells was analyzed using electric cell-substrate impedance sensing (ECIS) and scratch wound assay. We then used a microscope to evaluate the cell spread. We assessed filamentous actin (F-actin) cytoskeleton formation through immunofluorescence staining. In addition, we used Western blot analysis to analyze the expression of phospho-focal adhesion kinase (FAK), FAK, phospho-paxillin, paxillin, and F-actin. We discovered that lidocaine had an inhibitory effect on the migration of tenocytes in the scratch wound assay and on the ECIS chip. Lidocaine treatment suppressed cell spreading and changed the cell morphology and F-actin distribution. Lidocaine reduced F-actin formation in the tenocyte during cell spreading; furthermore, it inhibited phospho-FAK, F-actin, and phospho-paxillin expression in the tenocytes. Our study revealed that lidocaine inhibits the spread and migration of tenocytes. The molecular mechanism potentially underlying this effect is downregulation of F-actin, phospho-FAK, and phospho-paxillin expression when cells are treated with lidocaine.

Extraciliary OFD1 Is Involved in Melanocyte Survival through Cell Adhesion to ECM via Paxillin.

Primary cilia play a significant role in influencing cell fate, including apoptosis in multiple cell types. In the lesional epidermis of vitiligo patients, a reduced number of ciliated cells was observed. Our study also revealed a downregulation of oral-facial digital syndrome type 1 (OFD1) in the affected skin of vitiligo patients. However, it remains unknown whether primary cilia are involved in the control of melanocyte apoptosis. While both intraflagellar transport 88 (IFT88) and retinitis pigmentosa GTPase regulator-interacting protein-1 like (RPGRIP1L) are associated with ciliogenesis in melanocytes, only the knockdown of OFD1, but not IFT88 and RPGRIP1L, resulted in increased melanocyte apoptosis. OFD1 knockdown led to a decrease in the expression of proteins involved in cell-extracellular matrix (ECM) interactions, including paxillin. The OFD1 amino acid residues 601-1012 interacted with paxillin, while the amino acid residues 1-601 were associated with ciliogenesis, suggesting that the OFD1 domains responsible for paxillin binding are distinct from those involved in ciliogenesis. OFD1 knockdown, but not IFT88 knockdown, inhibited melanocyte adhesion to the ECM, a defect that was restored by paxillin overexpression. In summary, our findings indicate that the downregulation of OFD1 induces melanocyte apoptosis, independent of any impairment in ciliogenesis, by reducing melanocyte adhesion to the ECM via paxillin.

Fyn-Mediated Paxillin Tyrosine 31 Phosphorylation Regulates Migration and Invasion of Breast Cancer Cells.

Metastasis is the leading cause of death in breast cancer patients due to the lack of effective therapies. Elevated levels of paxillin expression have been observed in various cancer types, with tyrosine phosphorylation shown to play a critical role in driving cancer cell migration. However, the specific impact of the distinct tyrosine phosphorylation events of paxillin in the progression of breast cancer remains to be fully elucidated. Here, we found that paxillin overexpression in breast cancer tissue is associated with a patient's poor prognosis. Paxillin knockdown inhibited the migration and invasion of breast cancer cells. Furthermore, the phosphorylation of paxillin tyrosine residue 31 (Tyr31) was significantly increased upon the TGF-ß1-induced migration and invasion of breast cancer cells. Inhibiting Fyn activity or silencing Fyn decreases paxillin Tyr31 phosphorylation. The wild-type and constitutively active Fyn directly phosphorylate paxillin Tyr31 in an in vitro system, indicating that Fyn directly phosphorylates paxillin Tyr31. Additionally, the non-phosphorylatable mutant of paxillin at Tyr31 reduces actin stress fiber formation, migration, and invasion of breast cancer cells. Taken together, our results provide direct evidence that Fyn-mediated paxillin Tyr31 phosphorylation is required for breast cancer migration and invasion, suggesting that targeting paxillin Tyr31 phosphorylation could be a potential therapeutic strategy for mitigating breast cancer metastasis.

An ULK1/2-PXN mechanotransduction pathway suppresses breast cancer cell migration.

The remodeling and stiffening of the extracellular matrix (ECM) is a well-recognized modulator of breast cancer progression. How changes in the mechanical properties of the ECM are converted into biochemical signals that direct tumor cell migration and metastasis remain poorly characterized. Here, we describe a new role for the autophagy-inducing serine/threonine kinases ULK1 and ULK2 in mechanotransduction. We show that ULK1/2 activity inhibits the assembly of actin stress fibers and focal adhesions (FAs) and as a consequence impedes cell contraction and migration, independent of its role in autophagy. Mechanistically, we identify PXN/paxillin, a key component of the mechanotransducing machinery, as a direct binding partner and substrate of ULK1/2. ULK-mediated phosphorylation of PXN at S32 and S119 weakens homotypic interactions and liquid-liquid phase separation of PXN, impairing FA assembly, which in turn alters the mechanical properties of breast cancer cells and their response to mechanical stimuli. ULK1/2 and the well-characterized PXN regulator, FAK/Src, have opposing functions on mechanotransduction and compete for phosphorylation of adjacent serine and tyrosine residues. Taken together, our study reveals ULK1/2 as important regulator of PXN-dependent mechanotransduction.

Evaluation of SgK269 expression in colon cancer patients and the effects of hAMSCs secretome on tumor invasion through SgK269/c-Src/p-P130Cas/p-Paxillin/p-ERK1/2 signaling pathway in HT-29 colon cancer cells.

Colon cancer is the fifth leading cause of cancer-related deaths worldwide. Stem cells have unique characteristics and are considered as a novel therapeutic platform for cancer. Sugen Kinase 269 (SgK269) is considered as an oncogenic scaffolding pseudo kinase which governs the rearranging of the cytoskeleton, cellular motility, and invasion. The aim of this study is to evaluate the expression of SgK269 in colon cancer patients and explore the therapeutic effects of human amniotic mesenchymal stromal cells (hAMSCs) on invasion and proliferation of colon cancer cells (HT-29) through analyzing SgK269/c-Src/p-P130Cas/p-Paxillin/p-ERK1/2 signaling pathway. In this regard, we collected 30 samples from colon cancer patients and evaluated SgK269 expression using quantitative real-time PCR (qRT-PCR). Next, we employed a co-culture system using Transwell 6-well plates and after 72 h, tumor growth promotion and invasion were analyzed in hAMSCs-treated HT-29 cells through SgK269/c-Src/p-P130Cas/p-Paxillin/p-ERK1/2/Rac signaling pathway using qRT-PCR, western blot method, MTT assay, wound healing assay, and DAPI staining. Our results showed upregulation of SgK269 in colon cancer patients. Treatment of HT-29 colon cancer cells with hAMSCs secretome can inhibit SgK269/c-Src/p-P130Cas/p-Paxillin/p-ERK1/2/Rac signaling pathway and the resulting suppression of cell invasion and proliferation. Our results suggest that SgK269 is an important target in colon cancer therapy and MSCs secretome may be an effective therapeutic approach to inhibit colon cancer cell invasion and proliferation through SgK269/c-Src/p-P130Cas/p-Paxillin/p-ERK1/2/Rac signaling pathway.

Paxillin interactome identified by SILAC and label-free approaches coupled to TurboID sheds light on the compositions of focal adhesions in mouse embryonic stem cells.

Self-renewal and differentiation of mouse embryonic stem cells (mESCs) are greatly affected by the extracellular matrix (ECM) environment; the composition and stiffness of which are sensed by the cells via integrin-associated focal adhesions (FAs) which link the cells to the ECM. Although FAs have been studied extensively in differentiated cells, their composition and function in mESCs are not as well elucidated. To gain more detailed knowledge of the molecular compositions of FAs in mESCs, we adopted the proximity-dependent biotinylation (BioID) proteomics approach. Paxillin, a known FA protein (FAP), is fused to the promiscuous biotin ligase TurboID as bait. We employed both SILAC- and label-free (LF)-based quantitative proteomics to strengthen as well as complement individual approach. The mass spectrometry data derived from SILAC and LF identified 38 and 443 proteins, respectively, with 35 overlapping candidates. Fifteen of these shared proteins are known FAPs based on literature-curated adhesome and 7 others are among the reported "meta-adhesome", suggesting the components of FAs are largely conserved between mESCs and differentiated cells. Furthermore, the LF data set contained an additional 18 literature-curated FAPs. Notably, the overlapped proteomics data failed to detect LIM-domain proteins such as zyxin family proteins, which suggests that FAs in mESCs are less mature than differentiated cells. Using the LF approach, we are able to identify PDLIM7, a LIM-domain protein, as a FAP in mESCs. This study illustrates the effectiveness of TurboID in mESCs. Importantly, we found that application of both SILAC and LF methods in combination allowed us to analyze the TurboID proteomics data in an unbiased, stringent and yet comprehensive manner.

Equine ß-defensin 1 regulates cytokine expression and phagocytosis in S. aureus-infected mouse monocyte macrophages via the Paxillin-FAK-PI3K pathway.

ß-defensin-1 (BD-1) is a rich source of disulfide bonds and antibacterial peptides that exhibit direct bactericidal function. The expression of BD-1 is primarily induced by external stimulation and is known to correlate with TLR-mediated inflammation, suggesting its association with innate immune responses. Equine ß-defensin-1 (eBD-1) belongs to the BD-1 family. Our previous study demonstrated that eBD-1 enhances cytokine expression and promotes macrophage phagocytosis of S. aureus, although the underlying mechanism remains unknown. In this study, we utilized a PI-3K inhibitor (PKI-402) to treat eBD-1 -treated S. aureus-infected macrophages in vitro. Our results revealed that PKI-402 decreased the expression of eBD-1-promoted TNF-α, IL-6, CXCL10, CD40, RANTES, and p65 mRNA. To further investigate the relationship between eBD-1 and phagocytosis, we examined the expression of paxillin and FcγRIII (CD16 receptor) using western blot and immunofluorescence techniques. Our findings demonstrated that eBD-1 enhanced CD16 and paxillin expression in S. aureus -infected macrophages. Considering the correlation between paxillin expression and focal adhesion kinase (FAK), we transfected FAK siRNA into macrophages and evaluated paxillin expression using western blot analysis. Additionally, we quantified the number of S. aureus phagocytosed by macrophages. The results indicated a reduction in both paxillin expression and the number of S. aureus phagocytosed by macrophages upon FAK siRNA treatment. Our study showed the eBD-1 promotes cytokine mRNA expression in S. aureus-infected macrophages regulated by PI-3K-NF-κB pathway, and it increases macrophage phagocytosis of S. aureus associated with the FAK-paxillin signaling pathway.

Paxillin knockout in mouse granulosa cells increases fecundity†.

Paxillin is an intracellular adaptor protein involved in focal adhesions, cell response to stress, steroid signaling, and apoptosis in reproductive tissues. To investigate the role of paxillin in granulosa cells, we created a granulosa-specific paxillin knockout mouse model using Cre recombinase driven by the Anti-Müllerian hormone receptor 2 gene promoter. Female granulosa-specific paxillin knockout mice demonstrated increased fertility in later reproductive age, resulting in higher number of offspring when bred continuously up to 26 weeks of age. This was not due to increased numbers of estrous cycles, ovulated oocytes per cycle, or pups per litter, but this was due to shorter time to pregnancy and increased number of litters in the granulosa-specific paxillin knockout mice. The number of ovarian follicles was not significantly affected by the knockout at 30 weeks of age. Granulosa-specific paxillin knockout mice had slightly altered estrous cycles but no difference in circulating reproductive hormone levels. Knockout of paxillin using clustered regularly interspaced short palindromic repeat-associated protein 9 (CRISPR-Cas9) in human granulosa-derived immortalized KGN cells did not affect cell proliferation or migration. However, in cultured primary mouse granulosa cells, paxillin knockout reduced cell death under basal culture conditions. We conclude that paxillin knockout in granulosa cells increases female fecundity in older reproductive age mice, possibly by reducing granulosa cell death. This study implicates paxillin and its signaling network as potential granulosa cell targets in the management of age-related subfertility.

Evaluation of Paxillin Expression in Epithelial Dysplasia, Oral Squamous Cell Carcinoma, Lichen Planus with and without Dysplasia, and Hyperkeratosis: A Retrospective Cross-Sectional Study.

BACKGROUND: Paxillin is a cytoskeletal protein involved in the pathogenesis of several types of cancers. However, the roles of paxillin in epithelial dysplasia (ED), oral squamous cell carcinoma (OSCC), oral lichen planus with dysplasia (OLPD), hyperkeratosis (HK), and oral lichen planus (OLP) have remained unnoticed in the literature. This study aimed to evaluate its attainable functions in the pathogenesis and malignant transformation of potentially malignant oral epithelium and benign lesions. METHODS: In this retrospective cross-sectional study, paxillin expression was investigated in 99 tissue samples, including 18 cases of OSCC, 21 ED, 23 OLP, 21 OLPD, and 16 cases of HK. The tissue sections also underwent immunohistochemical paxillin staining using 3,3-diaminobenzidine (DAB) chromogen. The intensity, location, and percentage of staining were examined across all groups. Data were analyzed using the Shapiro-Wilk test, ANOVA, Pearson chi-square, Kruskal-Wallis, and Dunn's post hoc test. RESULTS: The cytoplasmic percentage and intensity staining of Paxillin expression were evident in the central/suprabasal and basal/peripheral layers of all the obtained samples. The final staining score was significantly higher in OSCC and dysplasia compared to HK and OLP (p = 0.004). It was found that paxillin expression is associated with the grade of dysplastic samples (p < 0.001). CONCLUSION: The present study provides evidence that paxillin may be involved in the pathogenesis of OSCC and the development and progression of dysplastic tissue, since the paxillin expression was higher than that of HK and OLP.

Direct active Fyn-paxillin interaction regulates vascular smooth muscle cell migration.

Vascular smooth muscle cell (VSMC) migration plays an important role in cardiovascular diseases, including atherosclerotic plaque formation and restenosis after vascular intervention. The mechanisms involved in VSMC migration are complex and have not been fully elucidated. Recently, we discovered a novel interaction, direct binding of active Fyn-paxillin at focal adhesions, which plays an important role in actin stress fiber formation and migration in VSMCs. In this review, we highlight paxillin as an intermediate signaling molecule that mediates actin stress fiber formation and VSMC migration through the Fyn/paxillin/Rho-kinase signaling pathway by directly binding to active Fyn. We also discuss the inhibition of VSMC migration by blocking the active Fyn-paxillin interaction and the potential of this interaction as a therapeutic target for cardiovascular diseases.