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.

Testis-specific serine kinase 6 (TSSK6) is abnormally expressed in colorectal cancer and promotes oncogenic behaviors.

Cancer testis antigens (CTAs) are a collection of proteins whose expression is normally restricted to the gamete but abnormally activated in a wide variety of tumors. The CTA, Testis-specific serine kinase 6 (TSSK6), is essential for male fertility in mice. The functional relevance of TSSK6 to cancer, if any, has not previously been investigated. Here we find that TSSK6 is frequently anomalously expressed in colorectal cancer and patients with elevated TSSK6 expression have reduced relapse-free survival. Depletion of TSSK6 from colorectal cancer cells attenuates anchorage-independent growth, invasion, and growth in vivo. Conversely, overexpression of TSSK6 enhances anchorage independence and invasion in vitro as well as in vivo tumor growth. Notably, ectopic expression of TSSK6 in semi-transformed human colonic epithelial cells is sufficient to confer anchorage independence and enhance invasion. In somatic cells, TSSK6 co-localizes with and enhances the formation of paxillin and tensin-positive foci at the cell periphery, suggesting a function in focal adhesion formation. Importantly, TSSK6 kinase activity is essential to induce these tumorigenic behaviors. Our findings establish that TSSK6 exhibits oncogenic activity when abnormally expressed in colorectal cancer cells. Thus, TSSK6 is a previously unrecognized intervention target for therapy, which could exhibit an exceptionally broad therapeutic window.

Phosphatidylinositol 4-Kinase III Alpha Governs Cytoskeletal Organization for Invasiveness of Liver Cancer Cells.

BACKGROUND & AIMS: High expression of phosphatidylinositol 4-kinase III alpha (PI4KIIIα) correlates with poor survival rates in patients with hepatocellular carcinoma. In addition, hepatitis C virus (HCV) infections activate PI4KIIIα and contribute to hepatocellular carcinoma progression. We aimed at mechanistically understanding the impact of PI4KIIIα on the progression of liver cancer and the potential contribution of HCV in this process. METHODS: Several hepatic cell culture and mouse models were used to study the functional importance of PI4KIIIα on liver pathogenesis. Antibody arrays, gene silencing, and PI4KIIIα-specific inhibitor were applied to identify the involved signaling pathways. The contribution of HCV was examined by using HCV infection or overexpression of its nonstructural protein. RESULTS: High PI4KIIIα expression and/or activity induced cytoskeletal rearrangements via increased phosphorylation of paxillin and cofilin. This led to morphologic alterations and higher migratory and invasive properties of liver cancer cells. We further identified the liver-specific lipid kinase phosphatidylinositol 3-kinase C2 domain-containing subunit gamma (PIK3C2γ) working downstream of PI4KIIIα in regulation of the cytoskeleton. PIK3C2γ generates plasma membrane phosphatidylinositol 3,4-bisphosphate-enriched, invadopodia-like structures that regulate cytoskeletal reorganization by promoting Akt2 phosphorylation. CONCLUSIONS: PI4KIIIα regulates cytoskeleton organization via PIK3C2γ/Akt2/paxillin-cofilin to favor migration and invasion of liver cancer cells. These findings provide mechanistic insight into the contribution of PI4KIIIα and HCV to the progression of liver cancer and identify promising targets for therapeutic intervention.

Plexin D1 negatively regulates macrophage-derived foam cell migration via the focal adhesion kinase/Paxillin pathway.

BACKGROUND: Macrophage-derived foam cell formation is a hallmark of atherosclerosis and is retained during plaque formation. Strategies to inhibit the accumulation of these cells hold promise as viable options for treating atherosclerosis. Plexin D1 (PLXND1), a member of the Plexin family, has elevated expression in atherosclerotic plaques and correlates with cell migration; however, its role in macrophages remains unclear. We hypothesize that the guidance receptor PLXND1 negatively regulating macrophage mobility to promote the progression of atherosclerosis. METHODS: We utilized a mouse model of atherosclerosis based on a high-fat diet and an ox-LDL- induced foam cell model to assess PLXND1 levels and their impact on cell migration. Through western blotting, Transwell assays, and immunofluorescence staining, we explored the potential mechanism by which PLXND1 mediates foam cell motility in atherosclerosis. RESULTS: Our study identifies a critical role for PLXND1 in atherosclerosis plaques and in a low-migration capacity foam cell model induced by ox-LDL. In the aortic sinus plaques of ApoE-/- mice, immunofluorescence staining revealed significant upregulation of PLXND1 and Sema3E, with colocalization in macrophages. In macrophages treated with ox-LDL, increased expression of PLXND1 led to reduced pseudopodia formation and decreased migratory capacity. PLXND1 is involved in regulating macrophage migration by modulating the phosphorylation levels of FAK/Paxillin and downstream CDC42/PAK. Additionally, FAK inhibitors counteract the ox-LDL-induced migration suppression by modulating the phosphorylation states of FAK, Paxillin and their downstream effectors CDC42 and PAK. CONCLUSION: Our findings indicate that PLXND1 plays a role in regulating macrophage migration by modulating the phosphorylation levels of FAK/Paxillin and downstream CDC42/PAK to promoting atherosclerosis.

Paxillin regulates androgen receptor expression associated with granulosa cell focal adhesions.

Paxillin is a ubiquitously expressed adaptor protein integral to focal adhesions, cell motility, and apoptosis. Paxillin has also recently been implicated as a mediator of nongenomic androgen receptor (AR) signaling in prostate cancer and other cells. We sought to investigate the relationship between paxillin and AR in granulosa cells (GCs), where androgen actions, apoptosis, and focal adhesions are of known importance, but where the role of paxillin is understudied. We recently showed that paxillin knockout in mouse GCs increases fertility in older mice. Here, we demonstrate that paxillin knockdown in human granulosa-derived KGN cells, as well as knockout in mouse primary GCs, results in reduced AR protein but not reduced mRNA expression. Further, we find that both AR protein and mRNA half-lives are reduced by approximately one-third in the absence of paxillin, but that cells adapt to chronic loss of paxillin by upregulating AR gene expression. Using co-immunofluorescence and proximity ligation assays, we show that paxillin and AR co-localize at the plasma membrane in GCs in a focal adhesion kinase-dependent way, and that disruption of focal adhesions leads to reduced AR protein level. Our findings suggest that paxillin recruits AR to the GC membrane, where it may be sequestered from proteasomal degradation and poised for nongenomic signaling, as reported in other tissues. To investigate the physiological significance of this in disorders of androgen excess, we tested the effect of GC-specific paxillin knockout in a mouse model of polycystic ovary syndrome (PCOS) induced by chronic postnatal dihydrotestosterone (DHT) exposure. While none of the control mice had estrous cycles, 33% of paxillin knockout mice were cycling, indicating that paxillin deletion may offer partial protection from the negative effects of androgen excess by reducing AR expression. Paxillin-knockout GCs from mice with DHT-induced PCOS also produced more estradiol than GCs from littermate controls. Thus, paxillin may be a novel target in the management of androgen-related disorders in women, such as PCOS.

LncRNA MALAT1 facilitates BM-MSCs differentiation into endothelial cells and ameliorates erectile dysfunction via the miR-206/CDC42/PAK1/paxillin signalling axis.

BACKGROUND: Erectile dysfunction (ED) is a common male sexual dysfunction, with an increasing incidence, and the current treatment is often ineffective. METHODS: Vascular endothelial growth factor (VEGFA) was used to treat bone marrow-derived mesenchymal stem cells (BM-MSCs), and their cell migration rates were determined by Transwell assays. The expression of the von Willebrand Factor (vWF)VE-cadherin, and endothelial nitric oxide synthase(eNOS) endothelial markers was determined by qRT‒PCR and Western blot analyses. The MALAT1-induced differentiation of BM-MCs to ECs via the CDC42/PAK1/paxillin pathway was explored by transfecting VEGFA-induced BM-MSC with si-MALAT1 and overexpressing CDC42 and PAK1. The binding capacity between CDC42, PAK1, and paxillin in VEGFA-treated and non-VEGFA-treated BM-MSCs was examined by protein immunoprecipitation. MiR-206 was overexpressed in VEGFA-induced BM-MSC, and the binding sites of MALAT1, miR-206, and CDC42 were identified using a luciferase assay. Sixty male Sprague‒Dawley rats were divided into six groups (n = 10/group). DMED modelling was demonstrated by APO experiments and was assessed by measuring blood glucose levels. Erectile function was assessed by measuring the intracavernosa pressure (ICP) and mean arterial pressure (MAP). Penile erectile tissue was analysed by qRT‒PCR, Western blot analysis, and immunohistochemical staining. RESULTS: MALAT1 under VEGFA treatment conditions regulates the differentiation of BM-MSCs into ECs by modulating the CDC42/PAK1/paxillin axis. In vitro experiments demonstrated that interference with CDC42 and MALAT1 expression inhibited the differentiation of BM-MSCs to ECs. CDC42 binds to PAK1, and PAK1 binds to paxillin. In addition, CDC42 in the VEGFA group had a greater ability to bind to PAK1, whereas PAK1 in the VEGFA group had a greater ability to bind to paxillin. Overexpression of miR-206 in VEGFA-induced BM-MSCs demonstrated that MALAT1 competes with the CDC42 3'-UTR for binding to miR-206, which in turn is involved in the differentiation of BM-MSCs to ECs. Compared to the DMED model group, the ICP/MAP ratio was significantly greater in the three BM-MSCs treatment groups. CONCLUSIONS: MALAT1 facilitates BM-MSC differentiation into ECs by regulating the miR-206/CDC42/PAK1/paxillin axis to improve ED. The present findings revealed the vital role of MALAT1 in the repair of BM-MSCs for erectile function and provided new mechanistic insights into the BM-MSC-mediated repair of DMED.

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.

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.

Erucin, a natural isothiocyanate, exerts pro-angiogenic properties in cultured endothelial cells and reverts angiogenic impairment induced by high glucose.

Insufficient vessel maintenance adversely impacts patients in terms of tissue reperfusion following stroke or myocardial infarction, as well as during wound healing. Angiogenesis impairment is a feature typical of metabolic disorders acting at the cardiovascular level, such as diabetes. Therapeutic angiogenesis regulation offers promising clinical implications, and natural compounds as pro-angiogenic nutraceuticals hold valuable applications in regenerative medicine. By using cultured endothelial cells from human umbilical veins (HUVEC) we studied functional and molecular responses following exposure to erucin, a natural isothiocyanate derived from Brassicaceae plants and extracted from the seeds of rocket. Erucin (at nanomolar concentrations) promotes cell migration and tube formation, similar to vascular endothelial growth factor (VEGF), through mobilizing paxillin at endothelial edges. At the molecular level, erucin induces signaling pathways typical of angiogenesis activation, namely Ras, PI3K/AKT, and ERK1/2, leading to VEGF expression and triggering its autocrine production, as pharmacological inhibition of soluble VEGF and VEGFR2 dampens endothelial functions. Furthermore, erucin, alone and together with VEGF, preserves endothelial angiogenic functions under pathological conditions, such as those induced in HUVEC by high glucose (HG) exposure. Erucin emerges as a compelling candidate for therapeutic revascularization applications, showcasing promising prospects for natural compounds in regenerative medicine, particularly in addressing angiogenesis-related disorders.

Gallic acid attenuates metastatic potential of human colorectal cancer cells through the miR-1247-3p-modulated integrin/FAK axis.

Colorectal cancer (CRC) exhibits highly metastatic potential even in the early stages of tumor progression. Gallic acid (GA), a common phenolic compound in plants, is known to possess potent antioxidant and anticancer activities, thereby inducing cell death or cell cycle arrest. However, whether GA reduces the invasiveness of CRC cells without inducing cell death remains unclear. Herein, we aimed to investigate the antimetastatic activity of low-dose GA on CRC cells and determine its underlying mechanism. Cell viability and tumorigenicity were analyzed by MTS, cell adhesion, and colony formation assay. Invasiveness was demonstrated using migration and invasion assays. Changes in protein phosphorylation and expression were assessed by Western blot. The involvement of microRNAs was validated by microarray analysis and anti-miR antagonist. Our findings showed that lower dose of GA (≤100 µM) did not affect cell viability but reduced the capabilities of colony formation, cell adhesion, and invasiveness in CRC cells. Cellularly, GA downregulated the cellular level of integrin αV/ß3, talin-1, and tensin and diminished the phosphorylated FAK, paxillin, Src, and AKT in DLD-1 cells. Microarray results revealed that GA increased miR-1247-3p expression, and pretreatment of anti-miR antagonist against miR-1247-3p restored the GA-reduced integrin αV/ß3 and the GA-inhibited paxillin activation in DLD-1 cells. Consistently, the in vivo xenograft model showed that GA administration inhibited tumor growth and liver metastasis derived from DLD-1 cells. Collectively, our findings indicated that GA inhibited the metastatic capabilities of CRC cells, which may result from the suppression of integrin/FAK axis mediated by miR1247-3p.

Yohimbine Inhibits PDGF-Induced Vascular Smooth Muscle Cell Proliferation and Migration via FOXO3a Factor.

Yohimbine (YHB) has been reported to possess anti-inflammatory, anticancer, and cardiac function-enhancing properties. Additionally, it has been reported to inhibit the proliferation, migration, and neointimal formation of vascular smooth muscle cells (VSMCs) induced by platelet-derived growth factor (PDGF) stimulation by suppressing the phospholipase C-gamma 1 pathway. However, the transcriptional regulatory mechanism of YHB controlling the behavior of VSMCs is not fully understood. In this study, YHB downregulated the expression of cell cycle regulatory proteins, such as proliferating cell nuclear antigen (PCNA), cyclin D1, cyclin-dependent kinase 4 (CDK4), and cyclin E, by modulating the transcription factor FOXO3a in VSMCs induced by PDGF. Furthermore, YHB decreased p-38 and mTOR phosphorylation in a dose-dependent manner. Notably, YHB significantly reduced the phosphorylation at Y397 and Y925 sites of focal adhesion kinase (FAK), and this effect was greater at the Y925 site than Y397. In addition, the expression of paxillin, a FAK-associated protein known to bind to the Y925 site of FAK, was significantly reduced by YHB treatment in a dose-dependent manner. A pronounced reduction in the migration and proliferation of VSMCs was observed following co-treatment of YHB with mTOR or p38 inhibitors. In conclusion, this study shows that YHB inhibits the PDGF-induced proliferation and migration of VSMCs by regulating the transcription factor FOXO3a and the mTOR/p38/FAK signaling pathway. Therefore, YHB may be a potential therapeutic candidate for preventing and treating cardiovascular diseases such as atherosclerosis and vascular restenosis.

[The influence of Ras-associated binding protein 23 knockdown on the migration and invasion of esophageal squamous cell carcinoma cells and its mechanism].

Objective: To investigate the role and the mechanism of Ras-associated binding protein23 (RAB23) in the migration and invasion of esophageal squamous cell carcinoma (ESCC) cells. Methods: RAB23 mRNA levels were measured in 16 pairs of ESCC and adjacent normal tissues via real-time polymerase chain reactions. RAB23 mRNA levels in the ESCC and adjacent normal tissues of dataset GSE20347 deposited in the Gene Expression Omnibus (GEO) database were also analyzed. Immunohistochemistry (IHC) was used to detect the RAB23 protein expressions in 106 pairs of ESCC and adjacent normal tissues, as well as in the lymph glands and primary tumor tissues of 33 patients with positive lymph nodes and 10 patients with negative lymph nodes. Endogenous RAB23 expression was transiently depleted using siRNAs (si-NC, si-RAB23-1, and si-RAB23-9) or stably reduced using shRNAs (sh-NC and sh-RAB23) in ESCC KYSE30 and KYSE150 cells, and the knockdown efficiency was tested using Western blot assays. Cell counting kit-8 assays and mouse xenograft models were used to test the proliferation of ESCC cells. Transwell assays and tail vein-pulmonary metastasis models in immunocompromised mice were used to examine the migration and invasion of ESCC cells. Cell adhesion assays were used to test the adhesion of ESCC cells. RNA-seq assays were used to analyze how RAB23 knockdown influenced the expression profile of ESCC cells and the implicated signal pathways were confirmed using Western blot assays. Results: The RAB23 mRNA expression in 16 cases of ESCC tissues was 0.009 7±0.008 9, which was markedly higher than that in adjacent normal tissues (0.003 2±0.003 7, P=0.006). GEO analysis on RAB23 expressions in ESCC and adjacent normal tissues showed that the RAB23 mRNA level in ESCC tissues (4.30±0.25) was remarkably increased compared with their normal counterparts (4.10±0.17, P=0.037). Among the 106 pairs of ESCC and tumor-adjacent normal tissues, 51 cases exhibited low expression of RAB23 and 55 cases showed high expression of RAB23, whereas in the paired tumor-adjacent normal tissues 82 cases were stained weakly and 24 strongly for RAB23 protein. These results indicated that RAB23 expression was markedly increased in ESCC tissues (P<0.001). Additionally, only 1 out of 33 primary ESCC tissues with positive lymph nodes showed low RAB23 protein expression. On the other hand, 7 samples of primary ESCC tissues with negative lymph nodes were stained strongly for RAB23 while its level in the other 3 samples was weak. These results showed that RAB23 expression was remarkably increased in primary ESCC tissues with positive lymph nodes compared with those with negative lymph nodes (P=0.024). Further tests showed that 32 out of 33 positive lymph nodes were stained strongly for RAB23, whereas no negative lymph nodes (n=10) exhibited high expression of RAB23 (P<0.001). Both transient and stable knockdown of endogenous RAB23 expression failed to cause detectable changes in the proliferation of KYSE30 cells in vitro and in vivo, but attenuated the migration and invasion of KYSE30 cells as well as the invasion of KYSE150 cells. RAB23 knockdown was found to significantly decrease the number of adhesive KYSE30 cells in the sh-RAB23 group (313.75±89.34) compared with control cells in the sh-NC group (1 030.75±134.29, P<0.001). RAB23 knockdown was also found to significantly decrease the number of adhesive KYSE150 cells in the sh-RAB23 group (710.5±31.74) compared with the number of control cells in the sh-NC group (1 005.75±61.09, P<0.001). RNA-seq assays demonstrated that RAB23 knockdown using two siRNAs targeting RAB23 mRNA markedly impaired focal adhesion-related signal pathways, and decreased the levels of phosphorylated FAK (p-FAK) and phosphorylated paxillin (p-paxillin) in KYSE30 and KYSE150 cells. Conclusions: Significantly increased RAB23 in ESCC tissues positively correlates with lymph node metastasis. Depleted RAB23 expression attenuates focal adhesion-related signal pathways, thus impairing the invasion, metastasis, and adhesion of ESCC cells.

Evaluation of the levels of salivary paxillin in oral potentially malignant disorders and malignant lesions.

BACKGROUND: The scientific community has been particularly interested in oral squamous cell carcinoma (OSCC) because of the cancer's extremely high incidence and fatality rates worldwide. It has been proposed that paxillin is involved in certain malignancies as an oncogene. Additionally, several investigations have assessed paxillin expression and investigated its function in developing distinct human carcinomas, including squamous cell carcinoma. Furthermore, it was discovered that there is a strong link between paxillin levels and cancer progression and spread. OBJECTIVE: This investigation was carried out to analyze and compare the salivary paxillin levels between oral potentially malignant disorders (OPMDs), OSCC and the healthy controls to assess its potential role as a biomarker of oral cancer aiming for early diagnosis and better prognosis of OSCC. METHODS: Forty-five patients, ranging in age from thirty to seventy-five, were divided into three groups: fifteen patients with OPMDs, fifteen patients with OSCC, and fifteen controls. Paxillin was identified in saliva samples by using an ELISA kit. RESULTS: Patients with OSCC and OPMDs have considerably greater salivary Paxillin levels than the healthy control group. The receiver operating curve (ROC) analysis was used in our study to distinguish patients with OPMDs from those with OSCC. The ROC curve constructed with the OPMDs group as the positives had lower sensitivity and area under the curve (AUC) values [100% and 1] than the ROC curve with the malignant group as the positives [93.3% and 0.997], respectively. Furthermore, ROC analysis performed between OPMDs group and the malignant group showed a specificity of 73.3% and a cut-off value ≥ 7.9 . CONCLUSION: Paxillin can be considered a reliable biomarker for identifying and comparing OPMDs and OSCC cancerous changes. GOV IDENTIFIER: NCT06154551- 4/12/2023.

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.

[Lutein inhibits the adhesion, invasiveness and metastasis of human prostate cancer PC-3M cells].

OBJECTIVE: To explore the effects of lutein on the adhesion, invasiveness and metastasis of human prostate cancer PC-3M cells and its action mechanism. METHODS: We divided human prostate cancer PC-3M cells into a control, a low-dose lutein, a medium-dose lutein and a high-dose lutein group, and treated them with 0, 10, 20 and 40 µmol/L lutein, respectively. Then we examined the adhesion of the cells to matrix by cell adhesion assay and the changes in cell pseudopodia by Phalloidin staining, detected the expressions of paxillin, matrix metalloproteinase 2 (MMP-2), MMP-9, recombinant tissue inhibitors of metalloproteinase 1 (TIMP-1), E-cadherin, N-cadherin and vimentin by Western blot, determined the invasiveness and migration of the cells by scratch and Transwell assays, and observed their dynamic movement by high-intension imaging. RESULTS: Compared with the control, the lutein intervention groups showed significant reduction in the number of the cells adhered to matrix, the number of cell pseudopodia, the expressions of paxillin, MMP-2, MMP-9, N-cadherin and vimentin, the rates of migration, invasion and metastasis, and the distances of displacement and movement of the cells. However, the expressions of TIMP-1 and epithelial-mesenchymal transition-related E-cadherin were upregulated significantly. CONCLUSION: Lutein can inhibit cell adhesion, reduce the expressions of MMPs, and suppress cell invasion and migration by inhibiting the process of epithelial-mesenchymal transition.

Paxillin phase separation promotes focal adhesion assembly and integrin signaling.

Focal adhesions (FAs) are transmembrane protein assemblies mediating cell-matrix connection. Although protein liquid-liquid phase separation (LLPS) has been tied to the organization and dynamics of FAs, the underlying mechanisms remain unclear. Here, we experimentally tune the LLPS of PXN/Paxillin, an essential scaffold protein of FAs, by utilizing a light-inducible Cry2 system in different cell types. In addition to nucleating FA components, light-triggered PXN LLPS potently activates integrin signaling and subsequently accelerates cell spreading. In contrast to the homotypic interaction-driven LLPS of PXN in vitro, PXN condensates in cells are associated with the plasma membrane and modulated by actomyosin contraction and client proteins of FAs. Interestingly, non-specific weak intermolecular interactions synergize with specific molecular interactions to mediate the multicomponent condensation of PXN and are efficient in promoting FA assembly and integrin signaling. Thus, our data establish an active role of the PXN phase transition into a condensed membrane-associated compartment in promoting the assembly/maturation of FAs.

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.

Substrate Stiffness Modulates Stemness and Differentiation of Rabbit Corneal Endothelium Through the Paxillin-YAP Pathway.

Purpose: To explore the role of substrate stiffness and the mechanism beneath corneal endothelial cells' (CECs') stemness maintenance and differentiation. Methods: CECs were divided into central zone (8 mm trephined boundary) and peripheral zone (8 mm trephined edge with attached limbal). Two zones were analyzed by hematoxylin-eosin staining and scanning electron microscopy for anatomic structure. The elastic modulus of Descemet's membrane (DM) was analyzed by atomic force microscopy. Compressed type I collagen gels with different stiffness were constructed as an in vitro model system to test the role of stiffness on phenotype using cultured rabbit CECs. Cell morphology, expression and intracellular distribution of Yes-associated protein (YAP), differentiation (ZO-1, Na+/K+-ATPase), stemness (FOXD3, CD34, Sox2, Oct3/4), and endothelial-mesenchymal transition (EnMT) markers were analyzed by immunofluorescence, quantitative RT-PCR, and Western blot. Results: The results showed that the peripheral area of rabbit and human DM is softer than the central area ex vivo. Using the biomimetic extracellular matrix collagen gels in vitro model, we then demonstrated that soft substrate weakens the differentiation and EnMT in the culture of CECs. It was further proved by the inhibitor experiment that soft substrate enhances stemness maintenance via inhibition of paxillin-YAP signaling, which was activated on a stiff substrate. Conclusions: Our findings confirm that substrate stiffness modulates the stemness maintenance and differentiation of CECs and suggest a potential strategy for CEC-based corneal tissue engineering.

FOXM1 Participates in Scleral Remodeling in Myopia by Upregulating APOA1 Expression Through METTL3/YTHDF2.

Purpose: Apolipoprotein A1 (APOA1) is a potential crucial protein and treatment goal for pathological myopia in humans. This study set out to discover the function of APOA1 in scleral remodeling in myopia and its underlying mechanisms. Methods: A myopic cell model was induced using hypoxia. Following loss- and gain-of function experiments, the expression of the myofibroblast transdifferentiation-related and collagen production-related factors Forkhead box M1 (FOXM1), APOA1, and methyltransferase-like 3 (METTL3) in the myopic cell model was examined by quantitative reverse transcription polymerase chain reaction (RT-qPCR) and western blotting. The proliferation and apoptosis were determined by Cell Counting Kit-8 assay and flow cytometry, respectively. Chromatin immunoprecipitation (ChIP) was employed to examine FOXM1 enrichment in the METTL3 promoter, methylated RNA immunoprecipitation (Me-RIP) to examine the N6-methyladenosine (m6A) modification level of APOA1, and photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) to examine the binding between METTL3 and APOA1. Results: Hypoxia-induced human scleral fibroblasts (HSFs) had high APOA1 and FOXM1 expression and low METTL3 expression. FOXM1 knockdown elevated METTL3 expression and downregulated APOA1 expression. FOXM1 was enriched in METTL3 promoter. APOA1 or FOXM1 knockdown or METTL3 overexpression reversed the hypoxia-induced elevation in vinculin, paxillin, and α-smooth muscle actin (α-SMA) levels and apoptosis and the reduction in collagen, type I, alpha 1 (COL1A1) level and cell proliferation in HSFs. METTL3 or YTH N6-methyladenosine RNA binding protein F2 (YTHDF2) knockdown or APOA1 overexpression reversed the impacts of FOXM1 knockdown on vinculin, paxillin, α-SMA, and COL1A1 expression and cell proliferation and apoptosis. Conclusions: FOXM1 elevated the m6A methylation level of APOA1 by repressing METTL3 transcription and enhanced APOA1 mRNA stability and transcription by reducing the YTHDF2-recognized m6A methylated transcripts.

Rbfox1 controls alternative splicing of focal adhesion genes in cardiac muscle cells.

Alternative splicing is one of the major cellular processes that determine the tissue-specific expression of protein variants. However, it remains challenging to identify physiologically relevant and tissue-selective proteins that are generated by alternative splicing. Hence, we investigated the target spectrum of the splicing factor Rbfox1 in the cardiac muscle context in more detail. By using a combination of in silico target prediction and in-cell validation, we identified several focal adhesion proteins as alternative splicing targets of Rbfox1. We focused on the alternative splicing patterns of vinculin (metavinculin isoform) and paxillin (extended paxillin isoform) and identified both as potential Rbfox1 targets. Minigene analyses suggested that both isoforms are promoted by Rbfox1 due to binding in the introns. Focal adhesions play an important role in the cardiac muscle context, since they mainly influence cell shape, cytoskeletal organization, and cell-matrix association. Our data confirmed that depletion of Rbfox1 changed cardiomyoblast morphology, cytoskeletal organization, and multinuclearity after differentiation, which might be due to changes in alternative splicing of focal adhesion proteins. Hence, our results indicate that Rbfox1 promotes alternative splicing of focal adhesion genes in cardiac muscle cells, which might contribute to heart disease progression, where downregulation of Rbfox1 is frequently observed.