AB1952 Sigma-AldrichAnti-Integrin beta1 Antibody, Cytosolic

Steroids and growth factors control neuronal development through their receptors under physiological and pathological conditions. We show that PC12 cells harbor endogenous androgen receptor (AR), whose inhibition or silencing strongly interferes with neuritogenesis stimulated by the nonaromatizable synthetic androgen R1881 or NGF. This implies a role for AR not only in androgen signaling, but also in NGF signaling. In turn, a pharmacological TrkA inhibitor interferes with NGF- or androgen-induced neuritogenesis. In addition, androgen or NGF triggers AR association with TrkA, TrkA interaction with PI3-K δ, and downstream activation of PI3-K δ and Rac in PC12 cells. Once associated with AR, filamin A (FlnA) contributes to androgen or NGF neuritogenesis, likely through its interaction with signaling effectors, such as Rac. This study thus identifies a previously unrecognized reciprocal cross-talk between AR and TrkA, which is controlled by β1 integrin. The contribution of FlnA/AR complex and PI3-K δ to neuronal differentiation by androgens and NGF is also novel. This is the first description of AR function in PC12 cells.

Retinal ganglion cells (RGCs) are exposed to injury in a variety of optic nerve diseases including glaucoma. However, not all cells respond in the same way to damage and the capacity of individual RGCs to survive or regenerate is variable. In order to elucidate factors that may be important for RGC survival and regeneration we have focussed on the extracellular matrix (ECM) and RGC integrin expression. Our specific questions were: (1) Do adult RGCs express particular sets of integrins in vitro and in vivo? (2) Can the nature of the ECM influence the expression of different integrins? (3) Can the nature of the ECM affect the survival of the cells and the length or branching complexity of their neurites?Primary RGC cultures from adult rat retina were placed on glass coverslips treated with different substrates: Poly-L-Lysine (PL), or PL plus laminin (L), collagen I (CI), collagen IV (CIV) or fibronectin (F). After 10 days in culture, we performed double immunostaining with an antibody against βIII-Tubulin to identify the RGCs, and antibodies against the integrin subunits: αV, α1, α3, α5, β1 or β3. The number of adhering and surviving cells, the number and length of the neurites and the expression of the integrin subunits on the different substrates were analysed.PL and L were associated with the greatest survival of RGCs while CI provided the least favourable conditions. The type of substrate affected the number and length of neurites. L stimulated the longest growth. We found at least three different types of RGCs in terms of their capacity to regenerate and extend neurites. The different combinations of integrins expressed by the cells growing on different substrata suggest that RGCs expressed predominantly α1β1 or α3β1 on L, α1β1 on CI and CIV, and α5β3 on F. The activity of the integrins was demonstrated by the phosphorylation of focal adhesion kinase (FAK).Adult rat RGCs can survive and grow in the presence of different ECM tested. Further studies should be done to elucidate the different molecular characteristics of the RGCs subtypes in order to understand the possible different sensitivity of different RGCs to damage in diseases like glaucoma in which not all RGCs die at the same time.

The collecting system of the kidney develops from the ureteric bud (UB), which undergoes branching morphogenesis, a process regulated by multiple factors, including integrin-extracellular matrix interactions. The laminin (LM)-binding integrin α3β1 is crucial for this developmental program; however, the LM types and LM/integrin α3β1-dependent signaling pathways are poorly defined. We show that α3 chain-containing LMs promote normal UB branching morphogenesis and that LM-332 is a better substrate than LM-511 for stimulating integrin α3β1-dependent collecting duct cell functions. We demonstrate that integrin α3β1-mediated cell adhesion to LM-332 modulates Akt activation in the developing collecting system and that Akt activation is PI3K independent but requires decreased PTEN activity and K63-linked polyubiquitination. We identified the ubiquitin-modifying enzyme TRAF6 as an interactor with the integrin β1 subunit and regulator of integrin α3β1-dependent Akt activation. Finally, we established that the developmental defects of TRAF6- and integrin α3-null mouse kidneys are similar. Thus K63-linked polyubiquitination plays a previously unrecognized role in integrin α3β1-dependent cell signaling required for UB development and may represent a novel mechanism whereby integrins regulate signaling pathways.

Vascular permeability is frequently associated with inflammation and is triggered by a cohort of secreted permeability factors such as vascular endothelial growth factor (VEGF). Here, we show that the physiological vascular permeability that precedes implantation is directly controlled by progesterone receptor (PR) and is independent of VEGF. Global or endothelial-specific deletion of PR blocks physiological vascular permeability in the uterus, whereas misexpression of PR in the endothelium of other organs results in ectopic vascular leakage. Integration of an endothelial genome-wide transcriptional profile with chromatin immunoprecipitation sequencing revealed that PR induces an NR4A1 (Nur77/TR3)-dependent transcriptional program that broadly regulates vascular permeability in response to progesterone. Silencing of NR4A1 blocks PR-mediated permeability responses, indicating a direct link between PR and NR4A1. This program triggers concurrent suppression of several junctional proteins and leads to an effective, timely, and venous-specific regulation of vascular barrier function that is critical for embryo implantation.

Human essential hypertension is characterized by eutrophic inward remodeling of the resistance arteries with little evidence of hypertrophy. Upregulation of αVβ3 integrin is crucial during this process. In order to investigate the role of focal adhesion kinase (FAK) activation in this process, the level of FAK Y397 autophosphorylation was studied in small blood vessels from young TGR(mRen2)27 animals as blood pressure rose and eutrophic inward remodeling took place. Between weeks 4 and 5, this process was completed and accompanied by a significant increase in FAK phosphorylation compared with normotensive control animals. Phosphorylated (p)FAK Y397 was coimmunoprecipitated with both β1- and β3-integrin-specific antibodies. In contrast, only a fraction (less than 10-fold) was coprecipitated with the β3 integrin subunit in control vessels. Inhibition of eutrophic remodeling by cRGDfV treatment of TGR(mRen2)27 rats resulted in the development of smooth-muscle-cell hypertrophy and a significant further enhancement of FAK Y397 phosphorylation, but this time with exclusive coassociation of pFAK Y397 with integrin β1. We established that phosphorylation of FAK Y397 with association with β1 and β3 integrins occurs with pressure-induced eutrophic remodeling. Inhibiting this process leads to an adaptive hypertrophic vascular response induced by a distinct β1-mediated FAK phosphorylation pattern.

Activated chemokine receptor initiates inside-out signaling to transiently trigger activation of integrins, a process involving multiple components that have not been fully characterized. Here we report that GM-CSF/IL-3/IL-5 receptor common beta-chain-associated protein (CBAP) is required to optimize this inside-out signaling and activation of integrins. First, knockdown of CBAP expression in human Jurkat T cells caused attenuated CXC chemokine ligand-12 (CXCL12)-induced cell migration and integrin α4β1- and αLβ2-mediated cell adhesion in vitro, which could be rescued sufficiently upon expression of murine CBAP proteins. Freshly isolated CBAP-deficient primary T cells also exhibited diminution of chemotaxis toward CC chemokine ligand-21 (CCL21) and CXCL12, and these chemokines-induced T-cell adhesions in vitro. Adoptive transfer of isolated naive T cells demonstrated that CBAP deficiency significantly reduced lymph node homing ability in vivo. Finally, migration of T cell-receptor-activated T cells induced by inflammatory chemokines was also attenuated in CBAP-deficient cells. Further analyses revealed that CBAP constitutively associated with both integrin β1 and ZAP70 and that CBAP is required for chemokine-induced initial binding of the talin-Vav1 complex to integrin β1 and to facilitate subsequent ZAP70-mediated dissociation of the talin-Vav1 complex and Vav1 phosphorylation. Within such an integrin signaling complex, CBAP likely functions as an adaptor and ultimately leads to activation of both integrin α4β1 and Rac1. Taken together, our data suggest that CBAP indeed can function as a novel signaling component within the ZAP70/Vav1/talin complex and plays an important role in regulating chemokine-promoted T-cell trafficking.

Intercellular adhesion molecule-5 (ICAM-5) is a dendrite-specific adhesion molecule, which functions in both the immune and nervous systems. ICAM-5 is the only negative regulator that has been identified for maturation of dendritic spines so far. Shedding of the ICAM-5 ectodomain promotes spine maturation and enhances synaptic activity. However, the mechanism by which ICAM-5 regulates spine development remains poorly understood. In this study, we found that ablation of ICAM5 expression resulted in a significant increase in the formation of synaptic contacts and the frequency of miniature excitatory post-synaptic currents, an indicator of pre-synaptic release probability. Antibodies against ICAM-5 and β1 integrins altered spine maturation. Furthermore, we found that β1 integrins serve as binding partners for ICAM-5. β1 integrins were immunoprecipitated with ICAM-5 from mouse brain and the binding region in ICAM-5 was localized to the two first Ig domains. β1 integrins were juxtaposed to filopodia tips at the early stage of synaptic formation, but as synapses matured, β1 integrins covered the mushroom spines. Loss of β1 integrins from the pre-synaptic sites affected the morphology of the post-synaptic structures. ICAM-5 ectodomain cleavage decreased or increased when the interaction between ICAM-5 and β1 integrins was potentiated or weakened, respectively, using antibodies. These results suggest that the interaction between ICAM-5 and β1 integrins is important in formation of functional synapses.

The directional migration and the following development of primordial germ cells (PGCs) during gonad formation are key steps for germline development. It has been proposed that the interaction between germ cells and genital ridge (GR) somatic cells plays essential roles in this process. However, the in vivo functional requirements of GR somatic cells in germ cell development are largely unknown.Wt1 mutation (Wt1(R394W/R394W)) results in GR agenesis through mitotic arrest of coelomic epitheliums. In this study, we employed the GR-deficient mouse model, Wt1(R394W/R394W), to investigate the roles of GR somatic cells in PGC migration and proliferation. We found that the number of PGCs was dramatically reduced in GR-deficient embryos at embryonic day (E) 11.5 and E12.5 due to decreased proliferation of PGCs, involving low levels of BMP signaling. In contrast, the germ cells in Wt1(R394W/R394W) embryos were still mitotically active at E13.5, while all the germ cells in control embryos underwent mitotic arrest at this stage. Strikingly, the directional migration of PGCs was not affected by the absence of GR somatic cells. Most of the PGCs reached the mesenchyme under the coelomic epithelium at E10.5 and no ectopic PGCs were noted in GR-deficient embryos. However, the precise positioning of PGCs was disrupted.Our work provides in vivo evidence that the proliferation of germ cells is precisely regulated by GR somatic cells during different stages of gonad development. GR somatic cells are probably dispensable for the directional migration of PGCs, but they are required for precise positioning of PGCs at the final step of migration.

Although the pivotal role of platelet derived growth factor (PDGF)-mediated signaling in vascular diseases was demonstrated, the pathophysiological mechanisms driving its over-activation remain incompletely understood. Tissue transglutaminase (tTG) is a multifunctional protein expressed in the vasculature, including smooth muscle cells (SMCs), and implicated in several vascular pathologies. The goal of this study is to define the regulation of PDGF-BB/PDGFRβ-induced signaling pathways and cell responses by tTG in vascular SMCs. We find that in human aortic SMCs, shRNA-mediated depletion and over-expression of tTG reveals its ability to down-regulate PDGFRβ levels and induce receptor clustering. In these cells, tTG specifically amplifies the activation of PDGFRβ and its multiple downstream signaling targets in response to PDGF-BB. Furthermore, tTG promotes dedifferentiation and increases survival, proliferation, and migration of human aortic SMCs mediated by this growth factor. Finally, PDGF-BB stimulates tTG expression in human aortic SMCs in culture and in the blood vessels in response to injury. Together, our results show that tTG in vascular SMCs acts as a principal enhancer within the PDGF-BB/PDGFRβ signaling axis involved in phenotypic modulation of these cells, thereby suggesting a novel role for this protein in the progression of vascular diseases.

β1 integrin has been shown to contribute to vascular smooth muscle cell differentiation, adhesion and mechanosensation in vitro. Here we showed that deletion of β1 integrin at the onset of smooth muscle differentiation resulted in interrupted aortic arch, aneurysms and failure to assemble extracellular matrix proteins. These defects result in lethality prior to birth. Our data indicates that β1 integrin is not required for the acquisition, but it is essential for the maintenance of the smooth muscle cell phenotype, as levels of critical smooth muscle proteins are gradually reduced in mutant mice. Furthermore, while deposition of extracellular matrix was not affected, its structure was disrupted. Interestingly, defects in extracellular matrix and vascular wall assembly, were restricted to the aortic arch and its branches, compromising the brachiocephalic and carotid arteries and to the exclusion of the descending aorta. Additional analysis of β1 integrin in the pharyngeal arch smooth muscle progenitors was performed using wnt1Cre. Neural crest cells deleted for β1 integrin were able to migrate to the pharyngeal arches and associate with endothelial lined arteries; but exhibited vascular remodeling defects and early lethality. This work demonstrates that β1 integrin is dispensable for migration and initiation of the smooth muscle differentiation program, however, it is essential for remodeling of the pharyngeal arch arteries and for the assembly of the vessel wall of their derivatives. It further establishes a critical role of β1 integrin in the protection against aneurysms that is particularly confined to the ascending aorta and its branches.