Preclinical evaluation of protein synthesis inhibitor omacetaxine in pediatric brainstem gliomas.

Background: Diffuse intrinsic pontine gliomas (DIPGs) pose a significant challenge as a highly aggressive and currently incurable form of pediatric brain cancer, necessitating the development of novel therapeutic strategies. Omacetaxine, an FDA-approved protein synthesis inhibitor for treating certain hematological malignancies, was investigated for its potential antitumor effects against preclinical DIPG models. Methods: We employed primary DIPG cultures to study omacetaxine's cytotoxicity and its impact on colony formation. Annexin V staining and flow cytometry assessed apoptosis. Wound healing assays evaluated migration, while western blotting determined inhibition of oncogenic proteins. We tested omacetaxine's therapeutic efficacy in an orthotopic DIPG model and assessed brain penetration using mass spectrometry. Results: We found a pronounced cytotoxic activity of omacetaxine against DIPG neurospheres, with low IC50 values of approximately 20 nM. Omacetaxine exerted its anti-proliferative effect by inhibiting protein synthesis and the induction of apoptotic pathways, evidenced by significant elevated levels of cleaved caspase 3 and cleaved PARP, both key markers of apoptosis. Omacetaxine effectively targeted oncogenic players such as PDGFRα and PI3K without additional effects on the mTOR signaling pathway. Furthermore, our study revealed the inhibitory effects of omacetaxine on cell migration, and a significant reduction in integrin/FAK signaling, which plays a crucial role in tumor progression and metastasis. Conclusions: Despite these promising in vitro effects, omacetaxine's efficacy in an orthotopic DIPG model was limited due to inadequate penetration across the blood-brain barrier. As such, further research and advancements are crucial to improve the drug's brain penetration, thus enhancing its overall therapeutic potential.

Mechanosensitive hormone signaling promotes mammary progenitor expansion and breast cancer risk.

Tissue stem-progenitor cell frequency has been implicated in tumor risk and progression, but tissue-specific factors linking these associations remain ill-defined. We observed that stiff breast tissue from women with high mammographic density, who exhibit increased lifetime risk for breast cancer, associates with abundant stem-progenitor epithelial cells. Using genetically engineered mouse models of elevated integrin mechanosignaling and collagen density, syngeneic manipulations, and spheroid models, we determined that a stiff matrix and high mechanosignaling increase mammary epithelial stem-progenitor cell frequency and enhance tumor initiation in vivo. Augmented tissue mechanics expand stemness by potentiating extracellular signal-related kinase (ERK) activity to foster progesterone receptor-dependent RANK signaling. Consistently, we detected elevated phosphorylated ERK and progesterone receptors and increased levels of RANK signaling in stiff breast tissue from women with high mammographic density. The findings link fibrosis and mechanosignaling to stem-progenitor cell frequency and breast cancer risk and causally implicate epidermal growth factor receptor-ERK-dependent hormone signaling in this phenotype.

Single-cell RNA-sequencing analysis of aortic valve interstitial cells demonstrates the regulation of integrin signaling by nitric oxide.

Calcific aortic valve disease (CAVD) is an increasingly prevalent condition among the elderly population that is associated with significant morbidity and mortality. Insufficient understanding of the underlying disease mechanisms has hindered the development of pharmacologic therapies for CAVD. Recently, we described nitric oxide (NO) mediated S-nitrosylation as a novel mechanism for preventing the calcific process. We demonstrated that NO donor or an S-nitrosylating agent, S-nitrosoglutathione (GSNO), inhibits spontaneous calcification in porcine aortic valve interstitial cells (pAVICs) and this was supported by single-cell RNA sequencing (scRNAseq) that demonstrated NO donor and GSNO inhibited myofibroblast activation of pAVICs. Here, we investigated novel signaling pathways that are critical for the calcification of pAVICs that are altered by NO and GSNO by performing an in-depth analysis of the scRNA-seq dataset. Transcriptomic analysis revealed 1,247 differentially expressed genes in pAVICs after NO donor or GSNO treatment compared to untreated cells. Pathway-based analysis of the differentially expressed genes revealed an overrepresentation of the integrin signaling pathway, along with the Rho GTPase, Wnt, TGF-ß, and p53 signaling pathways. We demonstrate that ITGA8 and VCL, two of the identified genes from the integrin signaling pathway, which are known to regulate cell-extracellular matrix (ECM) communication and focal adhesion, were upregulated in both in vitro and in vivo calcific conditions. Reduced expression of these genes after treatment with NO donor suggests that NO inhibits calcification by targeting myofibroblast adhesion and ECM remodeling. In addition, withdrawal of NO donor after 3 days of exposure revealed that NO-mediated transcriptional and translational regulation is a transient event and requires continuous NO exposure to inhibit calcification. Overall, our data suggest that NO and S-nitrosylation regulate the integrin signaling pathway to maintain healthy cell-ECM interaction and prevent CAVD.

Integrin α6ß4 signals through DNA damage response pathway to sensitize breast cancer cells to cisplatin.

Integrin α6ß4 is highly expressed in triple negative breast cancer (TNBC) and drives its most aggressive traits; however, its impact on chemotherapeutic efficacy remains untested. We found that integrin α6ß4 signaling promoted sensitivity to cisplatin and carboplatin but not to other chemotherapies tested. Mechanistic investigations revealed that integrin α6ß4 stimulated the activation of ATM, p53, and 53BP1, which required the integrin ß4 signaling domain. Genetic manipulation of gene expression demonstrated that mutant p53 cooperated with integrin α6ß4 for cisplatin sensitivity and was necessary for downstream phosphorylation of 53BP1 and enhanced ATM activation. Additionally, we found that in response to cisplatin-induced DNA double strand break (DSB), integrin α6ß4 suppressed the homologous recombination (HR) activity and enhanced non-homologous end joining (NHEJ) repair activity. Finally, we discovered that integrin α6ß4 preferentially activated DNA-PK, facilitated DNA-PK-p53 and p53-53BP1 complex formation in response to cisplatin and required DNA-PK to enhance ATM, 53BP1 and p53 activation as well as cisplatin sensitivity. In summary, we discovered a novel function of integrin α6ß4 in promoting cisplatin sensitivity in TNBC through DNA damage response pathway.

MFAP4-Mediated Effects in Elastic Fiber Homeostasis, Integrin Signaling and Cancer, and Its Role in Teleost Fish.

Microfibrillar-associated protein 4 (MFAP4) is an extracellular matrix (ECM) protein belonging to the fibrinogen-related domain superfamily. MFAP4 is highly expressed in elastin-rich tissues such as lung, blood vessels and skin. MFAP4 is involved in organization of the ECM, regulating proper elastic fiber assembly. On the other hand, during pathology MFAP4 actively contributes to disease development and progression due to its interactions with RGD-dependent integrin receptors. Both tissue expression and circulating MFAP4 levels are associated with various disorders, including liver fibrosis and cancer. In other experimental models, such as teleost fish, MFAP4 appears to participate in host defense as a macrophage-specific innate immune molecule. The aim of this review is to summarize the accumulating evidence that indicates the importance of MFAP4 in homeostasis as well as pathological conditions, discuss its known biological functions with special focus on elastic fiber assembly, integrin signaling and cancer, as well as describe the reported functions of non-mammalian MFAP4 in fish. Overall, our work provides a comprehensive overview on the role of MFAP4 in health and disease.

Microfibrillar-associated protein 4 in health and disease.

Microfibrillar-associated protein 4 (MFAP4) is an extracellular matrix protein belonging to the fibrinogen-related domain family. It has been localized to elastic fiber-rich regions in several tissues including the arteries, lungs, heart and skin. MFAP4 binds collagen, fibrillins and tropoelastin and contributes to the process of microfibrillar assembly and maturation of elastic fibers. MFAP4 can also bind RGD-dependent integrins, predominantly αVß3 and αVß5 through its N-terminal RGD sequence, modulating cellular behavior. Circulating MFAP4 was suggested as a robust biomarker for hepatitis C virus- and alcoholic liver disease-related liver fibrosis, cardiovascular disorders and chronic obstructive pulmonary disease. In mice, MFAP4 seems to have a widely redundant role under homeostatic conditions, as global MFAP4 deficiency results in a mild pulmonary phenotype, causing emphysema-like airspace enlargement that progresses with age. However, emerging in vivo and in vitro data suggest that MFAP4 is actively involved in the pathogenesis of remodeling-associated diseases, including fibrosis, cardiovascular disorders, aging, asthma and cancer through activation of integrin-mediated signaling as well as by modulating TGF-ß pathway, thus supporting maladaptive matrix remodeling. This review summarizes the current knowledge about MFAP4 structure and localization, its mechanisms of action in disease-induced tissue remodeling as well as its potential role as a clinical biomarker.

Pinch2 regulates myelination in the mouse central nervous system.

The extensive morphological changes of oligodendrocytes during axon ensheathment and myelination involve assembly of the Ilk-Parvin-Pinch (IPP) heterotrimeric complex of proteins to relay essential mechanical and biochemical signals between integrins and the actin cytoskeleton. Binding of Pinch1 and Pinch2 isoforms to Ilk is mutually exclusive and allows the formation of distinct IPP complexes with specific signaling properties. Using tissue-specific conditional gene ablation in mice, we reveal an essential role for Pinch2 during central nervous system myelination. Unlike Pinch1 gene ablation, loss of Pinch2 in oligodendrocytes results in hypermyelination and in the formation of pathological myelin outfoldings in white matter regions. These structural changes concur with inhibition of Rho GTPase RhoA and Cdc42 activities and phenocopy aspects of myelin pathology observed in corresponding mouse mutants. We propose a dual role for Pinch2 in preventing an excess of myelin wraps through RhoA-dependent control of membrane growth and in fostering myelin stability via Cdc42-dependent organization of cytoskeletal septins. Together, these findings indicate that IPP complexes containing Pinch2 act as a crucial cell-autonomous molecular hub ensuring synchronous control of key signaling networks during developmental myelination.

Calcium- and Integrin-Binding Protein 2 (CIB2) in Physiology and Disease: Bright and Dark Sides.

Calcium- and integrin-binding protein 2 (CIB2) is a small EF-hand protein capable of binding Mg2+ and Ca2+ ions. While its biological function remains largely unclear, an increasing number of studies have shown that CIB2 is an essential component of the mechano-transduction machinery that operates in cochlear hair cells. Mutations in the gene encoding CIB2 have been associated with non-syndromic deafness. In addition to playing an important role in the physiology of hearing, CIB2 has been implicated in a multitude of very different processes, ranging from integrin signaling in platelets and skeletal muscle to autophagy, suggesting extensive functional plasticity. In this review, we summarize the current understanding of biochemical and biophysical properties of CIB2 and the biological roles that have been proposed for the protein in a variety of processes. We also highlight the many molecular aspects that remain unclarified and deserve further investigation.

Quantitative Proteomics Reveals Molecular Network Driving Stromal Cell Differentiation: Implications for Corneal Wound Healing.

The differentiation of keratocytes to fibroblasts and myofibroblasts is an essential requisite during corneal wound closure. The aim of this study is to uncover factors involved in differentiation-dependent alteration in the protein profile of human corneal stromal cells using quantitative proteomics. Human corneal fibroblasts were cultured and differentiated into keratocytes in serum-free media and myofibroblasts through treatment with TGF-ß. The protein cell lysates from the donors were tryptic and were digested and labeled using a 3-plex iTRAQ kit. The labeled peptides were subjected to LCMS analysis. Biological functional analysis revealed a set of crucial proteins involved in the differentiation of human corneal stromal cells which were found to be significantly enriched. The selected proteins were further validated by immunohistochemistry. Quantitative proteomics identified key differentially expressed proteins which are involved in cellular signaling pathways. Proteins involved in integrin signaling (Ras-RAP1b, TLN and FN) and SLIT-ROBO pathways (PFN1, CAPR1, PSMA5) as well as extracellular matrix proteins (SERPINH1, SPARC, ITGß1, CRTAP) showed enhanced expression in corneal fibroblasts and myofibroblasts compared to keratocytes, indicating their possible role in wound healing. Corneal stromal cell differentiation is associated with the activation of diverse molecular pathways critical for the repair of fibroblasts and myofibroblasts. Identified proteins such as profilin 1 and talin could play a tentative role in corneal healing and serve as a potential target to treat corneal fibrosis.

LGL1 binds to Integrin ß1 and inhibits downstream signaling to promote epithelial branching in the mammary gland.

Branching morphogenesis is a fundamental process by which organs in invertebrates and vertebrates form branches to expand their surface areas. The current dogma holds that directional cell migration determines where a new branch forms and thus patterns branching. Here, we asked whether mouse Lgl1, a homolog of the Drosophila tumor suppressor Lgl, regulates epithelial polarity in the mammary gland. Surprisingly, mammary glands lacking Lgl1 have normal epithelial polarity, but they form fewer branches. Moreover, we find that Lgl1 null epithelium is unable to directionally migrate, suggesting that migration is not essential for mammary epithelial branching as expected. We show that LGL1 binds to Integrin ß1 and inhibits its downstream signaling, and Integrin ß1 overexpression blocks epithelial migration, thus recapitulating the Lgl1 null phenotype. Altogether, we demonstrate that Lgl1 modulation of Integrin ß1 signaling is essential for directional migration and that epithelial branching in invertebrates and the mammary gland is fundamentally distinct.

Clinical study of endoscopic surgery through thoraco mammary approach in treatment of thyroid papillary carcinoma / 中华内分泌外科杂志

Objective:To investigate the effect of endoscopic thyroidectomy through breast milk approach in patients with papillary thyroid carcinoma and its influence on Wnt and integrin signaling pathways.Methods:A total of 136 patients diagnosed with papillary thyroid carcinoma in our hospital from Jul. 2018 to Mar. 2020 were selected and were hospitalized for surgical treatment. According to different surgical procedures, they were divided into a study group (68 cases) and a control group (68 cases) . The control group was treated with open thyroidectomy and the study group was treated with thoracoscopic thyroidectomy. The two groups were compared in terms of immune function [CD4+, CD8+ and CD4+/CD8+], and pain index [PGE2, IL-6, Cor and VAS score]. RT-PCR method was used to detect WNT1, β-catenin, GSK3β and integrin Signal pathway before and after surgery.Results:Three days after operation, compared with the control group, the study group had significantly higher CD4+ and CD4+/CD8+ levels [ (27.62±2.52) vs (24.63±2.67) , (0.66±0.18) vs (0.52±0.13) ], while the CD8+ level was significantly lower [ (41.62±3.54) vs (45.62±3.63) ] ( P<0.001) ; PGE2, IL-6, Cor, VAS of the study group were significantly lower than the control group [ (48.54±9.86) vs (57.21±8.12) , (5.13±0.71) vs (6.99±0.95) , (511.23±67.52) vs (633.12±71.47) , (1.26±0.56) vs (3.99±2.06) ] ( P<0.001) ; WNT1, β-catenin, GSK3β, integrin β1, FAK, Ras, and MAPK mRNA expression levels in the study group were significantly lower than those of the control group[ (1.79±0.15) vs (2.85±0.25) , (1.94±0.15) vs (2.64±0.24) , (2.13±0.19) vs (2.97±0.28) , (1.95±0.17) vs (2.58± 0.23) , (2.15±0.16) vs (2.87±0.22) , (1.95±0.18) vs (2.91±0.27) , (1.89±0.12) vs (2.87±0.31) ] ( P<0.001) . Conclusion:Endoscopic thyroidectomy through thoracolumbar approach can effectively reduce postoperative pain in patients with papillary thyroid cancer, have a smaller impact on immune function, and block the expression of Wnt and integrin signaling pathways to reduce tumor metastasis risk.

Integrin signaling gene alterations and outcomes of cancer patients receiving immune checkpoint inhibitors.

BACKGROUND: Immune evasion is a hallmark of cancer and is associated with resistance to PD-1/PD-L1 and CTLA-4 inhibitors. Several interactions between tumor and immune cells within the tumor microenvironment are effected through integrin signaling; however the latter has been underrecognized as a pathway that could have an impact on oncological outcomes after treatment with immune checkpoint inhibitors (ICIs). This study aimed to assess the clinical relevance of genomic alterations in the integrin signaling pathway in ICI-treated patients with advanced cancers. METHODS: Next generation sequencing (NGS) data from tumor samples of patients with advanced cancers treated with ICIs (anti-PD-1/PD-L1, anti-CTLA4 or both) were queried from four independent publicly available cohorts for mutations and structural variations in 72 integrin signaling pathway genes (Gene Set: GOBP_CELL_ADHESION_MEDIATED_BY_INTEGRIN). The Kaplan Meier method was used to assess the association between mutated and unmutated genes with overall (OS) and progression-free survival (PFS). All results were reported at the 0.05 significance level. RESULTS: The largest cohort included 1662 patients (discovery set) and comprised 350 non-small cell lung cancer (NSCLC), 321 melanoma, 214 bladder, 151 renal cell carcinoma (RCC), 138 head neck (HN), 126 esophageal/gastric (EG), 117 glioma, 110 colorectal (CRC), 90 cancer of unknown primary (CUP), and 45 breast cancer patients. ICI treatments included PD-1 or PD-L1 inhibitors (n=1256), anti-CTLA4 inhibitors (n=146) or both (n=260). 170 patients (10% of the entire cohort) harbored mutations in PIK3CG (6%), RET (3%), SYK (1.4%), LYN (1.4%), PTPN11 (1.3%), and CRKL (0.1%) genes. Presence of these mutations was more frequent in melanoma (18%), followed by CRC (14.5%), CUP (11%), and NSCLC (11%). Patients with mutated tumors experienced a significantly longer median OS (41 months) compared to those without alterations (16 months, log-rank P<0.001). The favorable prognostic value of PIK3CG, RET, SYK, LYN, PTPN11, and CRKL alterations was confirmed in three melanoma cohorts (validation set, n=212, P=0.024). Assessment of mutation status of these genes in a fourth cohort of NSCLC patients (n=75) revealed a predictive significance as well, with regard to PFS after treatment with ipilimumab and nivolumab combination (P=0.048). CONCLUSION: Mutations and/or structural variations in integrin signaling genes may have prognostic and predictive value in patients with metastatic malignancies who receive ICIs. Although confirmation in larger studies with concurrent investigation of underlying immunologic mechanisms is needed, these findings pose therapeutic implications for co-targeted approaches to overcome immune evasion and resistance.

Syndecans and Their Synstatins: Targeting an Organizer of Receptor Tyrosine Kinase Signaling at the Cell-Matrix Interface.

Receptor tyrosine kinases (RTKs) and integrin matrix receptors have well-established roles in tumor cell proliferation, invasion and survival, often functioning in a coordinated fashion at sites of cell-matrix adhesion. Central to this coordination are syndecans, another class of matrix receptor, that organize RTKs and integrins into functional units, relying on docking motifs in the syndecan extracellular domains to capture and localize RTKs (e.g., EGFR, IGF-1R, VEGFR2, HER2) and integrins (e.g., αvß3, αvß5, α4ß1, α3ß1, α6ß4) to sites of adhesion. Peptide mimetics of the docking motifs in the syndecans, called "synstatins", prevent assembly of these receptor complexes, block their signaling activities and are highly effective against tumor cell invasion and survival and angiogenesis. This review describes our current understanding of these four syndecan-coupled mechanisms and their inhibitory synstatins (SSTNIGF1R, SSTNVEGFR2, SSTNVLA-4, SSTNEGFR and SSTNHER2).

Fibronectin-Mediated Inflammatory Signaling Through Integrin α5 in Vascular Remodeling.

Background Adhesion of vascular endothelial cells to the underlying basement membrane potently modulates endothelial cells to cells' inflammatory activation. The normal basement membrane proteins laminin and collagen IV attenuate inflammatory signaling in part through integrin α2ß1. In contrast, fibronectin, the provisional matrix protein found in injured, remodeling or inflamed vessels, sensitizes endothelial cells to inflammatory stimuli through integrins α5ß1and and αvß3. A chimeric integrin in which the cytoplasmic domain of α5 is replaced with that of α2 pairs with ß1 and binds fibronectin but signals like α2ß1. Methods and Results Here, we examined mice in which integrin α5 is replaced with the α5/2 chimera, using the transverse aortic constriction and partial carotid ligation models of vessel remodeling. Following transverse aortic constriction and partial carotid ligation surgery, wild-type mice showed increased fibronectin deposition and expression of inflammatory markers, which were strongly attenuated in a5/2 mice. α5/2 mice also showed reduced artery wall hypertrophy in the transverse aortic constriction model and diminished inward remodeling in the partial carotid ligation model. Acute atherosclerosis after partial carotid ligation in hyperlipidemic ApoE-/- mice on a high fat diet was dramatically decreased in α5/2 mice. Conclusions Fibronectin and integrin α5 signaling is a key element of pathological vascular remodeling in acute models of both hypertension and disturbed flow. These results underscore the key role for integrin α5 signaling in pathological vascular remodeling associated with hypertension and atherosclerosis and support its potential as a therapeutic target.

Identification of fibronectin 1 as a candidate genetic modifier in a Col4a1 mutant mouse model of Gould syndrome.

Collagen type IV alpha 1 and alpha 2 (COL4A1 and COL4A2) are major components of almost all basement membranes. COL4A1 and COL4A2 mutations cause a multisystem disorder that can affect any organ but typically involves the cerebral vasculature, eyes, kidneys and skeletal muscles. In recent years, patient advocacy and family support groups have united under the name of Gould syndrome. The manifestations of Gould syndrome are highly variable, and animal studies suggest that allelic heterogeneity and genetic context contribute to the clinical variability. We previously characterized a mouse model of Gould syndrome caused by a Col4a1 mutation in which the severities of ocular anterior segment dysgenesis (ASD), myopathy and intracerebral hemorrhage (ICH) were dependent on genetic background. Here, we performed a genetic modifier screen to provide insight into the mechanisms contributing to Gould syndrome pathogenesis and identified a single locus [modifier of Gould syndrome 1 (MoGS1)] on Chromosome 1 that suppressed ASD. A separate screen showed that the same locus ameliorated myopathy. Interestingly, MoGS1 had no effect on ICH, suggesting that this phenotype could be mechanistically distinct. We refined the MoGS1 locus to a 4.3 Mb interval containing 18 protein-coding genes, including Fn1, which encodes the extracellular matrix component fibronectin 1. Molecular analysis showed that the MoGS1 locus increased Fn1 expression, raising the possibility that suppression is achieved through a compensatory extracellular mechanism. Furthermore, we found evidence of increased integrin-linked kinase levels and focal adhesion kinase phosphorylation in Col4a1 mutant mice that is partially restored by the MoGS1 locus, implicating the involvement of integrin signaling. Taken together, our results suggest that tissue-specific mechanistic heterogeneity contributes to the variable expressivity of Gould syndrome and that perturbations in integrin signaling may play a role in ocular and muscular manifestations.

The Multiple Roles of the Cytosolic Adapter Proteins ADAP, SKAP1 and SKAP2 for TCR/CD3 -Mediated Signaling Events.

T cells are the key players of the adaptive immune response. They coordinate the activation of other immune cells and kill malignant and virus-infected cells. For full activation T cells require at least two signals. Signal 1 is induced after recognition of MHC/peptide complexes presented on antigen presenting cells (APCs) by the clonotypic TCR (T-cell receptor)/CD3 complex whereas Signal 2 is mediated via the co-stimulatory receptor CD28, which binds to CD80/CD86 molecules that are present on APCs. These signaling events control the activation, proliferation and differentiation of T cells. In addition, triggering of the TCR/CD3 complex induces the activation of the integrin LFA-1 (leukocyte function associated antigen 1) leading to increased ligand binding (affinity regulation) and LFA-1 clustering (avidity regulation). This process is termed "inside-out signaling". Subsequently, ligand bound LFA-1 transmits a signal into the T cells ("outside-in signaling") which enhances T-cell interaction with APCs (adhesion), T-cell activation and T-cell proliferation. After triggering of signal transducing receptors, adapter proteins organize the proper processing of membrane proximal and intracellular signals as well as the activation of downstream effector molecules. Adapter proteins are molecules that lack enzymatic or transcriptional activity and are composed of protein-protein and protein-lipid interacting domains/motifs. They organize and assemble macromolecular complexes (signalosomes) in space and time. Here, we review recent findings regarding three cytosolic adapter proteins, ADAP (Adhesion and Degranulation-promoting Adapter Protein), SKAP1 and SKAP2 (Src Kinase Associated Protein 1 and 2) with respect to their role in TCR/CD3-mediated activation, proliferation and integrin regulation.

Across scales: novel insights into kidney health and disease by structural biology.

Over the past decades, structural biology methods such as X-ray crystallography and cryo-electron microscopy have been increasingly used to study protein functions, molecular interactions, physiological processes, and disease mechanisms. This review outlines a selection of structural biology methods, highlights recent examples of how structural analyses have contributed to a more profound understanding of the machinery of life, and gives a perspective on how these methods can be applied to investigate functions of kidney molecules and pathogenic mechanisms of renal diseases.

Integrin-Linked Kinase Links Integrin Activation to Invadopodia Function and Invasion via the p(T567)-Ezrin/NHERF1/NHE1 Pathway.

Tumor cell invasion depends largely on degradation of the extracellular matrix (ECM) by protease-rich structures called invadopodia, whose formation and activity requires the convergence of signaling pathways engaged in cell adhesion, actin assembly, membrane regulation and ECM proteolysis. It is known that ß1-integrin stimulates invadopodia function through an invadopodial p(T567)-ezrin/NHERF1/NHE1 signal complex that regulates NHE1-driven invadopodia proteolytic activity and invasion. However, the link between ß1-integrin and this signaling complex is unknown. In this study, in metastatic breast (MDA-MB-231) and prostate (PC-3) cancer cells, we report that integrin-linked kinase (ILK) integrates ß1-integrin with this signaling complex to regulate invadopodia activity and invasion. Proximity ligation assay experiments demonstrate that, in invadopodia, ILK associates with ß1-integrin, NHE1 and the scaffold proteins p(T567)-ezrin and NHERF1. Activation of ß1-integrin increased both invasion and invadopodia activity, which were specifically blocked by inhibition of either NHE1 or ILK. We conclude that ILK integrates ß1-integrin with the ECM proteolytic/invasion signal module to induce NHE1-driven invadopodial ECM proteolysis and cell invasion.

Molecular mechanisms of voiding dysfunction in a novel mouse model of acute urinary retention.

Acute urinary retention (AUR) is a common urological emergency and affects a significant patient population. The inability to eliminate urine may lead to permanent damage to the bladder's structure and functioning. However, we know little about the underlying molecular sequelae to the urine retention. To closely mirror the potential high pressures that patients with AUR could experience, we catheterized anesthetized female mice via the urethra and filled the bladder by pumping saline (25 µL/min) into the bladder lumen to 50 cm or 80 cm water pressure. A water column with designated height (50 or 80 cm) was then adjusted to maintain constant pressure in the bladder lumen for 30 minutes. Functional and morphological evaluations were performed from 0 to 24 hours after AUR treatment. Mice exhibited incontinence and overactivity with diminished voiding pressure. Significant injury was confirmed which revealed bladders with disrupted urothelial barrier, edematous lamina propria, and distorted muscle bundles. Bladder smooth muscle (BSM) from pressure-treated mice have significantly diminished contraction force, suggesting that bladder voiding dysfunction can be attributed to impaired BSM contractility. Indeed, dysregulation of acetylcholine and purinergic signaling pathways were demonstrated, indicating that reduced efficacy of these pathways contributes to impaired BSM contractility. Finally, altered expression of ß1-integrin and extracellular matrix mediated mechanotransduction pathways were detected, suggesting a profound remodeling process. These data demonstrated an easy to perform, quantifiable, and reproducible AUR mouse model, which mimics well the characteristics of human AUR patients, and our data generate new insights into the molecular mechanisms that occur following AUR.

Manipulation of Focal Adhesion Signaling by Pathogenic Microbes.

Focal adhesions (FAs) serve as dynamic signaling hubs within the cell. They connect intracellular actin to the extracellular matrix (ECM) and respond to environmental cues. In doing so, these structures facilitate important processes such as cell-ECM adhesion and migration. Pathogenic microbes often modify the host cell actin cytoskeleton in their pursuit of an ideal replicative niche or during invasion to facilitate uptake. As actin-interfacing structures, FA dynamics are also intimately tied to actin cytoskeletal organization. Indeed, exploitation of FAs is another avenue by which pathogenic microbes ensure their uptake, survival and dissemination. This is often achieved through the secretion of effector proteins which target specific protein components within the FA. Molecular mimicry of the leucine-aspartic acid (LD) motif or vinculin-binding domains (VBDs) commonly found within FA proteins is a common microbial strategy. Other effectors may induce post-translational modifications to FA proteins through the regulation of phosphorylation sites or proteolytic cleavage. In this review, we present an overview of the regulatory mechanisms governing host cell FAs, and provide examples of how pathogenic microbes have evolved to co-opt them to their own advantage. Recent technological advances pose exciting opportunities for delving deeper into the mechanistic details by which pathogenic microbes modify FAs.