Β1 Integrin Activation Research Articles

In Situ Regulation and Mechanisms of 3D Matrix Stiffness on the Activation and Reversion of Hepatic Stellate Cells.

Activated hepatic stellate cells (HSCs) is a key event in the progression of liver fibrosis. HSCs transdifferentiate into myofibroblasts and secrete large amounts of extracellular matrix, resulting in increased liver stiffness. It is difficult for platforms constructed in vitro to simulate the structure, composition, and stiffness of the 3D microenvironment of HSCs in vivo. Here, 3D scaffolds with different stiffness are constructed by decellularizing rat livers at different stages of fibrosis. The effects of matrix stiffness on the proliferation, activation, and reversion of HSCs are studied. The results demonstrate these scaffolds have good cytocompatibility. It is also found that the high stiffness can significantly promote the activation of HSCs, and this process is accompanied by the activation of integrin β1 as well as the nucleation and activation of Yes-associated protein (YAP). Moreover, the low stiffness of the scaffold can promote the reversion of activated HSCs, which is associated with cell apoptosis and accompanied by the inactivation of integrin β1 and YAP. These results suggest that YAP may be a potential therapeutic target for the treatment of liver fibrosis and the theoretical feasibility of inducing activated HSCs reversion to the resting state by regulating matrix stiffness of liver.

Free fatty acids stabilize integrin β1via S-nitrosylation to promote monocyte–endothelial adhesion

Hyperlipidemia characterized by high blood levels of free fatty acids (FFAs) is important for the progression of inflammatory cardiovascular diseases. Integrin β1 is a transmembrane receptor that drives various cellular functions, including differentiation, migration, and phagocytosis. However, the underlying mechanisms modifying integrin β1 protein and activity in mediating monocyte/macrophage adhesion to endothelium remain poorly understood. In this study, we demonstrated that integrin β1 protein underwent S-nitrosylation in response to nitrosative stress in macrophages. To examine the effect of elevated levels of FFA on the modulation of integrin β1 expression, we treated the macrophages with a combination of oleic acid and palmitic acid (2:1) and found that FFA activated inducible nitric oxide synthase/nitric oxide and increased the integrin β1 protein level without altering the mRNA level. FFA promoted integrin β1 S-nitrosylation via inducible nitric oxide synthase/nitric oxide and prevented its degradation by decreasing binding to E3 ubiquitin ligase c-Cbl. Furthermore, we found that increased integrin α4β1 heterodimerization resulted in monocyte/macrophage adhesion to endothelium. In conclusion, these results provided novel evidence that FFA-stimulated N--O stabilizes integrin β1via S-nitrosylation, favoring integrin α4β1 ligation to promote vascular inflammation.

The βI domain promotes active β1 integrin clustering into mature adhesion sites.

Modulation of integrin function is required in many physiological and pathological settings, such as angiogenesis and cancer. Integrin allosteric changes, clustering, and trafficking cooperate to regulate cell adhesion and motility on extracellular matrix proteins via mechanisms that are partly defined. By exploiting four monoclonal antibodies recognizing distinct conformational epitopes, we show that in endothelial cells (ECs), the extracellular βI domain, but not the hybrid or I-EGF2 domain of active β1 integrins, promotes their FAK-regulated clustering into tensin 1-containing fibrillar adhesions and impairs their endocytosis. In this regard, the βI domain-dependent clustering of active β1 integrins is necessary to favor fibronectin-elicited directional EC motility, which cannot be effectively promoted by β1 integrin conformational activation alone.

β1-Integrin blockade prevents podocyte injury in experimental models of minimal change disease

IntroductionActivation of the focal adhesion kinase (FAK) in podocytes is involved in the pathogenesis of minimal change disease (MCD), but the pathway leading to its activation in this disease is unknown. Here, we tested whether podocyte β1 integrin is the upstream modulator of FAK activation and podocyte injury in experimental models of MCD-like injury. MethodsWe used lipopolysaccharide (LPS) and MCD sera to induce MCD-like changes in vivo and in cultured human podocytes, respectively. We performed functional studies using specific β1 integrin inhibitors in vivo and in vitro, and integrated histological analysis, western blotting, and immunofluorescence to assess for morphological and molecular changes in podocytes. By ELISA, we measured serum LPS levels in 35 children with MCD or presumed MCD (idiopathic nephrotic syndrome [INS]) and in 18 healthy controls. ResultsLPS-injected mice showed morphological (foot process effacement, and normal appearing glomeruli on light microscopy) and molecular features (synaptopodin loss, nephrin mislocalization, FAK phosphorylation) characteristic of human MCD. Administration of a β1 integrin inhibitor to mice abrogated FAK phosphorylation, and ameliorated proteinuria and podocyte injury following LPS. Children with MCD/INS in relapse had higher serum LPS levels than controls. In cultured human podocytes, β1 integrin blockade prevented cytoskeletal rearrangements following exposure to MCD sera in relapse. ConclusionsPodocyte β1 integrin activation is an upstream mediator of FAK phosphorylation and podocyte injury in models of MCD-like injury.

The Interaction between hERG1 and β1 Integrins Modulates hERG1 Current in Different Pathological Cell Models.

Ion channels are implicated in various diseases, including cancer, in which they modulate different aspects of cancer progression. In particular, potassium channels are often aberrantly expressed in cancers, a major example being provided by hERG1. The latter is generally complexed with β1 integrin in tumour cells, and such a molecular complex represents a new druggable hub. The present study focuses on the characterization of the functional consequences of the interaction between hERG1 and β1 integrins on different substrates over time. To this purpose, we studied the interplay alteration on the plasma membrane through patch clamp techniques in a cellular model consisting of human embryonic kidney (HEK) cells stably transfected with hERG1 and in a cancer cell model consisting of SH-SY5Y neuroblastoma cells, endogenously expressing the channel. Cells were seeded on different substrates known to stimulate β1 integrins, such as fibronectin (FN) for HEK-hERG1 and laminin (LMN) for SH-SY5Y. In HEK cells stably overexpressing hERG1, we observed a hERG1 current density increase accompanied by Vrest hyperpolarization after cell seeding onto FN. Notably, a similar behaviour was shown by SH-SY5Y neuroblastoma cells plated onto LMN. Interestingly, we did not observe this phenomenon when plating the cells on substrates such as Bovine Serum Albumin (BSA) or Polylysine (PL), thus suggesting a crucial involvement of ECM proteins as well as of β1 integrin activation.

Spatiotemporal characteristics of P-selectin-induced β2 integrin activation of human neutrophils under flow.

Activation of integrins is crucial for recruitment of flowing leukocytes to inflammatory or injured vascular sites, but their spatiotemporal characteristics are incompletely understood. We discovered that β2-integrin activation over the entire surface of neutrophils on immobilized P-selectin occurred via mitogen-activated protein kinase (MAPK) or non-MAPK signaling with a minute-level timescale in a force-dependent manner. In flow, MAPK signaling required intracellular Ca2+ release to activate integrin within 2min. Integrin activation via non-MAPK signaling occurred first locally in the vicinity of ligated P-selectin glycoprotein ligand-1 (PSGL-1) within sub-seconds, and then over the entire cell surface within 1min in an extracellular Ca2+ influx-dependent manner. The transition from a local (but rapid) to global (but slow) activation mode was triggered by ligating the freshly activated integrin. Lipid rafts, moesin, actin, and talin were involved in non-MAPK signaling. Fluid loads had a slight effect on local integrin activation with a second-level timescale, but served as enhancers of global integrin activation.

β1 integrin monoclonal antibody treatment ameliorates cerebral cavernous malformations.

β1 integrins are important in blood vessel formation and function, finely tuning the adhesion of endothelial cells to each other and to the extracellular matrix. The role of integrins in the vascular disease, cerebral cavernous malformation (CCM) has yet to be explored in vivo. Endothelial loss of the gene KRIT1 leads to brain microvascular defects, resulting in debilitating and often fatal consequences. We tested administration of a monoclonal antibody that enforces the active β1 integrin conformation, (clone 9EG7), on a murine neonatal CCM mouse model, Krit1flox/flox ;Pdgfb-iCreERT2 (Krit1ECKO ), and on KRIT1-silenced human umbilical vein endothelial cells (HUVECs). In addition, endothelial deletion of the master regulator of integrin activation, Talin 1 (Tln1), in Krit1ECKO mice was performed to assess the effect of completely blocking endothelial integrin activation on CCM. Treatment with 9EG7 reduced lesion burden in the Krit1ECKO model and was accompanied by a strong reduction in the phosphorylation of the ROCK substrate, myosin light chain (pMLC), in both retina and brain endothelial cells. Treatment of KRIT1-silenced HUVECs with 9EG7 in vitro stabilized cell-cell junctions. Overnight treatment of HUVECs with 9EG7 resulted in significantly reduced total surface expression of β1 integrin, which was associated with reduced pMLC levels, supporting our in vivo findings. Genetic blockade of integrin activation by Tln1ECKO enhanced bleeding and did not reduce CCM lesion burden in Krit1ECKO mice. In sum, targeting β1 integrin with an activated-specific antibody reduces acute murine CCM lesion development, which we found to be associated with suppression of endothelial ROCK activity.

Synergistic regulation of microglia differentiation by CD93 and integrin β1 in the rat pneumococcal meningitis model

Streptococcus pneumoniae is the main bacterial pathogen of meningitis worldwide, which has a high mortality rate and survivors are prone to central nervous system (CNS) sequelae. In this regard, microglia activation has been associated with injury to the CNS. The aim of this study was to investigate the relationship between CD93, integrin β1, and microglia activation. In the rat pneumococcal meningitis model, we found significant increases of CD93 and integrin β1 expression and differentiation of M1 phenotype microglia. Furthermore, we showed in vitro siRNA-mediated downregulation of CD93 and integrin β1 expression after infecting highly aggressive proliferating immortalized (HAPI) microglia cells with S. pneumoniae. We observed differentiation of S. pneumonia-infected HAPI microglia cells to the M1 phenotype and significant release of soluble CD93 (sCD93) and integrin β1 expression. Complement C1q and metalloproteinases promoted sCD93 release. We also showed that downregulation of CD93 significantly reduced differentiation to M1 microglia and increased differentiation to M2 microglia. However, addition of recombinant CD93 may regulate microglia differentiation to the M1 phenotype. Furthermore, the downregulation of integrin β1 resulted in downregulation of the CD93 protein. In conclusion, interaction between integrin β1 and CD93 promotes differentiation of microglia to the M1 phenotype, increases the release of pro-inflammatory factors, and leads to nervous system injury in pneumococcal meningitis.

Caveolae couple mechanical stress to integrin recycling and activation.

Cells are subjected to multiple mechanical inputs throughout their lives. Their ability to detect these environmental cues is called mechanosensing, a process in which integrins play an important role. During cellular mechanosensing, plasma membrane (PM) tension is adjusted to mechanical stress through the buffering action of caveolae; however, little is known about the role of caveolae in early integrin mechanosensing regulation. Here, we show that Cav1KO fibroblasts increase adhesion to FN-coated beads when pulled with magnetic tweezers, as compared to wild type fibroblasts. This phenotype is Rho-independent and mainly derived from increased active β1-integrin content on the surface of Cav1KO fibroblasts. Florescence recovery after photobleaching analysis and endocytosis/recycling assays revealed that active β1-integrin is mostly endocytosed through the clathrin independent carrier/glycosylphosphatidyl inositol (GPI)-enriched endocytic compartment pathway and is more rapidly recycled to the PM in Cav1KO fibroblasts, in a Rab4 and PM tension-dependent manner. Moreover, the threshold for PM tension-driven β1-integrin activation is lower in Cav1KO mouse embryonic fibroblasts (MEFs) than in wild type MEFs, through a mechanism dependent on talin activity. Our findings suggest that caveolae couple mechanical stress to integrin cycling and activation, thereby regulating the early steps of the cellular mechanosensing response.

Novel insights into mouse models of ectopic proplatelet release

AbstractMature bone marrow (BM) megakaryocytes (MKs) produce platelets by extending proplatelets into sinusoidal blood vessels. Defects in this process can lead to thrombocytopenia and increased risk of bleeding. Mice lacking the actin-regulatory proteins Profilin 1 (PFN1), Wiskott–Aldrich Syndrome protein (WASp), Actin Related Protein 2/3 complex (Arp2/3), or adhesion and degranulation-promoting adapter protein (ADAP) display thrombocytopenia and ectopic release of (pro)platelet-like particles into the BM compartment, pointing to an important axis of actin-mediated directional proplatelet formation. The mechanism underlying ectopic release in these mice is still not completely understood. However, we hypothesized that similar functional defects account for this observation. We analyzed WASp-, ADAP-, PFN1-, and ARPC2-knockout mice to determine the role of actin reorganization and integrin activation in directional proplatelet formation. ADAP-, ARPC2-, and PFN1-deficient MKs displayed reduced adhesion to collagen, defective F-actin organization, and diminished β1-integrin activation. WASp-deficient MKs showed the strongest reduction in the adhesion assay of collagen and altered F-actin organization with reduced podosome formation. Our results indicate that ADAP, PFN1, WASp, and ARP2/3 are part of the same pathway that regulates polarization processes in MKs and directional proplatelet formation into BM sinusoids.

Nexinhib20 Inhibits Neutrophil Adhesion and β2 Integrin Activation by Antagonizing Rac-1-Guanosine 5'-Triphosphate Interaction.

Neutrophils are critical for mediating inflammatory responses. Inhibiting neutrophil recruitment is an attractive approach for preventing inflammatory injuries, including myocardial ischemia-reperfusion (I/R) injury, which exacerbates cardiomyocyte death after primary percutaneous coronary intervention in acute myocardial infarction. In this study, we found out that a neutrophil exocytosis inhibitor Nexinhib20 inhibits not only exocytosis but also neutrophil adhesion by limiting β2 integrin activation. Using a microfluidic chamber, we found that Nexinhib20 inhibited IL-8-induced β2 integrin-dependent human neutrophil adhesion under flow. Using a dynamic flow cytometry assay, we discovered that Nexinhib20 suppresses intracellular calcium flux and β2 integrin activation after IL-8 stimulation. Western blots of Ras-related C3 botulinum toxin substrate 1 (Rac-1)-GTP pull-down assays confirmed that Nexinhib20 inhibited Rac-1 activation in leukocytes. An in vitro competition assay showed that Nexinhib20 antagonized the binding of Rac-1 and GTP. Using a mouse model of myocardial I/R injury, Nexinhib20 administration after ischemia and before reperfusion significantly decreased neutrophil recruitment and infarct size. Our results highlight the translational potential of Nexinhib20 as a dual-functional neutrophil inhibitory drug to prevent myocardial I/R injury.

Antagonistic control of active surface integrins by myotubularin and phosphatidylinositol 3-kinase C2β in a myotubular myopathy model

X-linked centronuclear myopathy (XLCNM) is a severe human disease without existing therapies caused by mutations in the phosphoinositide 3-phosphatase MTM1. Loss of MTM1 function is associated with muscle fiber defects characterized by impaired localization of β-integrins and other components of focal adhesions. Here we show that defective focal adhesions and reduced active β-integrin surface levels in a cellular model of XLCNM are rescued by loss of phosphatidylinositiol 3-kinase C2β (PI3KC2β) function. Inactivation of the Mtm1 gene impaired myoblast differentiation into myotubes and resulted in reduced surface levels of active β1-integrins as well as corresponding defects in focal adhesions. These phenotypes were rescued by concomitant genetic loss of Pik3c2b or pharmacological inhibition of PI3KC2β activity. We further demonstrate that a hitherto unknown role of PI3KC2β in the endocytic trafficking of active β1-integrins rather than rescue of phosphatidylinositol 3-phosphate levels underlies the ability of Pik3c2b to act as a genetic modifier of cellular XLCNM phenotypes. Our findings reveal a crucial antagonistic function of MTM1 and PI3KC2β in the control of active β-integrin surface levels, thereby providing a molecular mechanism for the adhesion and myofiber defects observed in XLCNM. They further suggest specific pharmacological inhibition of PI3KC2β catalysis as a viable treatment option for XLCNM patients.

A positive mechanobiological feedback loop controls bistable switching of cardiac fibroblast phenotype

Cardiac fibrosis is associated with activation of cardiac fibroblasts (CFs), a pathological, phenotypic transition that is widely believed to be irreversible in the late stages of disease development. Sensing of a stiffened mechanical environment through regulation of integrin-based adhesion plaques and activation of the Piezo1 mechanosensitive ion channel is known to factor into this transition. Here, using integrated in vitro and in silico models, we discovered a mutually reinforcing, mechanical positive feedback loop between integrin β1 and Piezo1 activation that forms a bistable switch. The bistable switch is initiated by perturbations in matrix elastic modulus that amplify to trigger downstream signaling involving Ca2+ and YAP that, recursively, leads fibroblasts to further stiffen their environment. By simultaneously interfering with the newly identified mechanical positive feedback loop and modulating matrix elastic modulus, we reversed markers of phenotypical transition of CF, suggesting new therapeutic targets for fibrotic disease.

β1 Integrin induces adhesion and migration of human Th17 cells via Pyk2-dependent activation of P2X4 receptor.

Integrin-mediated T-cell adhesion and migration is a crucial step in immune response and autoimmune diseases. However, the underlying signalling mechanisms are not fully elucidated. In this study, we examined the implication of purinergic signalling, which has been associated with T-cell activation, in the adhesion and migration of human Th17 cells across fibronectin, a major matrix protein associated with inflammatory diseases. We showed that the adhesion of human Th17 cells to fibronectin induces, via β1 integrin, a sustained release of adenosine triphosphate (ATP) from the mitochondria through the pannexin-1 hemichannels. Inhibition of ATP release or its degradation with apyrase impaired the capacity of the cells to attach and migrate across fibronectin. Inhibition studies identified a major role for the purinergic receptor P2X4 in T-cell adhesion and migration but not for P2X7 or P2Y11 receptors. Blockade of P2X4 but not P2X7 or P2Y11 receptors reduced cell adhesion and migration by inhibiting activation of β1 integrins, which is essential for ligand binding. Furthermore, we found that β1 integrin-induced ATP release, P2X4 receptor transactivation, cell adhesion and migration were dependent on the focal adhesion kinase Pyk2 but not FAK. Finally, P2X4 receptor inhibition also blocked fibronectin-induced Pyk2 activation suggesting the existence of a positive feedback loop of activation between β1 integrin/Pyk2 and P2X4 purinergic signalling pathways. Our findings uncovered an unrecognized link between β1 integrin and P2X4 receptor signalling pathways for promoting T-cell adhesion and migration across the extracellular matrix.

Cutaneous manifestations in Costello syndrome: HRAS p.Gly12Ser affects RIN1-mediated integrin trafficking in immortalized epidermal keratinocytes.

Heterozygous germline missense variants in the HRAS gene underlie Costello syndrome (CS). The molecular basis for cutaneous manifestations in CS is largely unknown. We used an immortalized human cell line, HaCaT keratinocytes, stably expressing wild-type or CS-associated (p.Gly12Ser) HRAS and defined RIN1 as quantitatively most prominent, high-affinity effector of active HRAS in these cells. As an exchange factor for RAB5 GTPases, RIN1 is involved in endosomal sorting of cell-adhesion integrins. RIN1-dependent RAB5A activation was strongly increased by HRASGly12Ser, and HRAS-RIN1-ABL1/2 signaling was induced in HRASWT- and HRASGly12Ser-expressing cells. Along with that, HRASGly12Ser expression decreased total integrin levels and enriched β1 integrin in RAB5- and EEA1-positive early endosomes. The intracellular level of active β1 integrin was increased in HRASGly12Ser HaCaT keratinocytes due to impaired recycling, whereas RIN1 disruption raised β1 integrin cell surface distribution. HRASGly12Ser induced co-localization of β1 integrin with SNX17 and RAB7 in early/sorting and late endosomes, respectively. Thus, by retaining β1 integrin in intracellular endosomal compartments, HRAS-RIN1 signaling affects the subcellular availability of β1 integrin. This may interfere with integrin-dependent processes as we detected for HRASGly12Ser cells spreading on fibronectin. We conclude that dysregulation of receptor trafficking and integrin-dependent processes such as cell adhesion are relevant in the pathobiology of CS.

CD146 interaction with integrin β1 activates LATS1-YAP signaling and induces radiation-resistance in breast cancer cells

Radiotherapy is an indispensable modality in comprehensive treatment of breast cancer. However, inherent or acquired radiation resistance of tumors compromises the efficacy of radiotherapy. Herein, we found that CD146, a unique epithelial-to-mesenchymal transition (EMT) inducer particularly highly expressed in triple-negative breast cancer (TNBC), is dramatically induced by ionizing irradiation. Further study demonstrates that CD146 promotes tumor cell radioresistance in vitro and in vivo. Specifically, we report the underlying mechanism that CD146 activates YAP protein, and drives its relocation from plasma to nucleus by regulating LATS1, and promoting abnormal DNA damage repair, as well as inducing EMT and stemness. Moreover, CD146 can form a novel co-receptor complex with integrin β1 and induces radiation-resistance in breast cancer. Dual inhibition of CD146 and integrin β1 activity had a stronger inhibitory effect on breast cancer tumor growth and synergistically increased their sensitivity to radiotherapy. This study identifies a unique function of CD146 implicates with integrin β1 and YAP signaling, contributing to radiation resistance. Targeted therapy against CD146 or inhibition of integrin β1 is a potential strategy to overcome radiotherapeutic resistance of breast cancer.

Graphene Oxide Loaded on TiO2-Nanotube-Modified Ti Regulates the Behavior of Human Gingival Fibroblasts.

Surface topography, protein adsorption, and the loading of coating materials can affect soft tissue sealing. Graphene oxide (GO) is a promising candidate for improving material surface functionalization to facilitate soft tissue integration between cells and biomaterials. In this study, TiO2 nanotubes (TNTs) were prepared by the anodization of Ti, and TNT-graphene oxide composites (TNT-GO) were prepared by subsequent electroplating. The aim of this study was to investigate the effect of TNTs and TNT-GO surface modifications on the behavior of human gingival fibroblasts (HGFs). Commercially pure Ti and TNTs were used as the control group, and the TNT-GO surface was used as the experimental group. Scanning electron microscopy, X-ray photoelectron spectroscopy, and X-ray diffraction were used to perform sample characterization. Cell adhesion, cell proliferation, cell immunofluorescence staining, a wound-healing assay, real-time reverse-transcriptase polymerase chain reaction (RT-PCR), and Western blotting showed that the proliferation, adhesion, migration, and adhesion-related relative gene expression of HGFs on TNT-GO were significantly enhanced compared to the control groups, which may be mediated by the activation of integrin β1 and the MAPK-Erk1/2 pathway. Our findings suggest that the biological reactivity of HGFs can be enhanced by the TNT-GO surface, thereby improving the soft tissue sealing ability.

Heterotropic roles of divalent cations in the establishment of allostery and affinity maturation of integrin αXβ2.

SUMMARYAllosteric activation and silencing of leukocyte β2-integrins transpire through cation-dependent structural changes, which mediate integrin biosynthesis and recycling, and are essential to designing leukocyte-specific drugs. Stepwise addition of Mg2+ reveals two mutually coupled events for the αXβ2 ligand-binding domain—the αX I-domain—corresponding to allostery establishment and affinity maturation. Electrostatic alterations in the Mg2+-binding site establish long-range couplings, leading to both pH− and Mg2+-occupancy-dependent biphasic stability change in the αX I-domain fold. The ligand-binding sensorgrams show composite affinity events for the αX I-domain accounting for the multiplicity of the αX I-domain conformational states existing in the solution. On cell surfaces, increasing Mg2+ concentration enhanced adhesiveness of αXβ2. This work highlights how intrinsically flexible pH− and cation-sensitive architecture endows a unique dynamic continuum to the αI-domain structure on the intact integrin, thereby revealing the importance of allostery establishment and affinity maturation in both extracellular and intracellular integrin events.

Abstract 6125: ADAM9 secreted by cancer-associated stromal cells promotes metastasis of prostate cancer

Abstract Introduction: Cancer-associated fibroblasts (CAFs), the main cellular component of the tumor microenvironment (TME), contribute to tumor metastasis. Prostate cancer (PCa) is the most common malignancy among the elderly male population, and it is typically fatal when the disease progresses to metastasis and castration-resistant stage (CRPC). We have previously demonstrated that ADAM9 increased during PCa progression, and ROS is the critical mediator regulating ADAM9 expression in CRPC cells to promote tumor growth. In addition to membrane-anchored ADAM9, a secreted variant of ADAM9 (sADAM9) was recently identified in activated liver stromal cells that facilitated colon cancer liver metastasis. However, it is not clear whether sADAM9 is expressed by prostatic CAFs and, if so, whether it contributes to aggressive tumor phenotypes through tumor-stromal interactions. Objective: To investigate the sADAM9 variants in CAFs and to elucidate the effects of CAF-derived sADAM9 on the aggressiveness of PCa cells. Methods: Immunohistochemistry staining and ELISA of clinical samples were used to assess the expression of ADAM9 during PCa tumor progression. Laser microdissected tissues, primary cell lines, and experimental cell model of paired normal fibroblasts and CAF cells were analyzed. The effects of sADAM9 on CRPC progression were conducted in PC3 cells by WST-1 cell proliferation assay, transwell migration assay, and μ-slide chemotaxis assay. Immunoblot analyses of integrin β1 and migration-related kinases were used to verify the possible downstream signaling pathway of sADAM9. Results: Increased overall intensity of ADAM9 protein expression in PCa patients with metastasis compared. Elevated ADAM9 expression was observed in both cancerous epithelial cells and the tumor stroma. The average serum ADAM9 concentration measured by ELISA was 6.253 ± 0.448 ng/mL in BPH patient samples versus 10.89 ± 0.953 ng/mL in PCa patient samples. A smaller molecular weight (~50-kDa) of ADAM9 was detected in CM from cultured CAFs but not normal fibroblasts or PCa cell lines except for DU145 cells. Treatment of PC3 cells with sADAM9 induced a significant chemotactic migration while did not affect cell proliferation. Furthermore, a stark increase in activated integrin β1, phosphorylated focal adhesion kinase (p-FAK), and p-AKT, but not p-mTOR in PCa cells by sADAM9 suggested the induction of cell migration occurs through activation of the integrin β1/FAK signaling pathways. Conclusion: Herein, we have provided in vitro evidence to demonstrate the pro-metastatic function of sADAM9 from CAFs that facilitate the migration of CRPC cells through integrin receptor signaling, which could be partly responsible for the promotion of CRPC metastasis. Further molecular identification of controlling spliced ADAM9 expression in CAFs remains to investigate. Citation Format: Weichieh Chen, Le Thi Huynh Trang, Chia-Ling Hsieh, Katsumi Shigemura, Masato Fujisawa, Leland W.K. Chung, Shian-Ying Sung. ADAM9 secreted by cancer-associated stromal cells promotes metastasis of prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6125.

Inner Nuclear Membrane Protein, SUN1, is Required for Cytoskeletal Force Generation and Focal Adhesion Maturation.

The linker of nucleoskeleton and cytoskeleton (LINC) complex is composed of the inner nuclear membrane-spanning SUN proteins and the outer nuclear membrane-spanning nesprin proteins. The LINC complex physically connects the nucleus and plasma membrane via the actin cytoskeleton to perform diverse functions including mechanotransduction from the extracellular environment to the nucleus. Mammalian somatic cells express two principal SUN proteins, namely SUN1 and SUN2. We have previously reported that SUN1, but not SUN2, is essential for directional cell migration; however, the underlying mechanism remains elusive. Because the balance between adhesive force and traction force is critical for cell migration, in the present study, we focused on focal adhesions (FAs) and the actin cytoskeleton. We observed that siRNA-mediated SUN1 depletion did not affect the recruitment of integrin β1, one of the ubiquitously expressed focal adhesion molecules, to the plasma membrane. Consistently, SUN1-depleted cells normally adhered to extracellular matrix proteins, including collagen, fibronectin, laminin, and vitronectin. In contrast, SUN1 depletion reduced the activation of integrin β1. Strikingly, the depletion of SUN1 interfered with the incorporation of vinculin into the focal adhesions, whereas no significant differences in the expression of vinculin were observed between wild-type and SUN1-depleted cells. In addition, SUN1 depletion suppressed the recruitment of zyxin to nascent focal adhesions. These data indicate that SUN1 is involved in the maturation of focal adhesions. Moreover, disruption of the SUN1-containing LINC complex abrogates the actin cytoskeleton and generation of intracellular traction force, despite the presence of SUN2. Thus, a physical link between the nucleus and cytoskeleton through SUN1 is required for the proper organization of actin, thereby suppressing the incorporation of vinculin and zyxin into focal adhesions and the activation of integrin β1, both of which are dependent on traction force. This study provides insights into a previously unappreciated signaling pathway from the nucleus to the cytoskeleton, which is in the opposite direction to the well-known mechanotransduction pathways from the extracellular matrix to the nucleus.