Integrins modulate fast excitatory transmission at hippocampal synapses

The present study provides the first evidence that adhesion receptors belonging to the integrin family modulate excitatory transmission in the adult rat brain. Infusion of an integrin ligand (the peptide GRGDSP) into rat hippocampal slices reversibly increased the slope and amplitude of excitatory postsynaptic potentials. This effect was not accompanied by changes in paired pulse facilitation, a test for perturbations to transmitter release, or affected by suppression of inhibitory responses, suggesting by exclusion that alterations to alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)-type glutamate receptors cause the enhanced responses. A mixture of function-blocking antibodies to integrin subunits alpha(3), alpha(5), and alpha(v) blocked ligand effects on synaptic responses. The ligand-induced increases were (i) blocked by inhibitors of Src tyrosine kinase, antagonists of N-methyl-d-aspartate receptors, and inhibitors of calcium calmodulin-dependent protein kinase II and (ii) accompanied by phosphorylation of both the Thr(286) site on calmodulin-dependent protein kinase II and the Ser(831) site on the GluR1 subunit of the AMPA receptor. N-Methyl-d-aspartate receptor antagonists blocked the latter two phosphorylation events, but Src kinase inhibitors did not. These results point to the conclusion that synaptic integrins regulate glutamatergic transmission and suggest that they do this by activating two signaling pathways directed at AMPA receptors.     

Integrin signaling cascades are operational in adult hippocampal synapses and modulate NMDA receptor physiology

Integrin class adhesion proteins are concentrated at adult brain synapses. Whether synaptic integrins engage kinase signaling cascades has not been determined, but is a question of importance to ideas about integrin involvement in functional synaptic plasticity. Accordingly, synaptoneurosomes from adult rat brain were used to test if matrix ligands activate integrin-associated tyrosine kinases, and if integrin signaling targets include NMDA-class glutamate neurotransmitter receptors. The integrin ligand peptide Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP) induced rapid (within 5 min) and robust increases in tyrosine phosphorylation of focal adhesion kinase, proline-rich tyrosine kinase 2 and Src family kinases. Increases were similarly induced by the native ligand fibronectin, blocked with neutralizing antibodies to beta1 integrin, and not obtained with control peptides, indicating that kinase activation was integrin-mediated. Both GRGDSP and fibronectin caused rapid Src kinase-dependent increases in tyrosine phosphorylation of NMDA receptor subunits NR2A and NR2B in synaptoneurosomes and acute hippocampal slices. Tests of the physiological significance of the latter result showed that ligand treatment caused a rapid and beta1 integrin-dependent increase in NMDA receptor-mediated synaptic responses. These results provide the first evidence that, in adult brain, synaptic integrins activate local kinase cascades with potent effects on the operation of nearby neurotransmitter receptors implicated in synaptic plasticity.     

Roles of glutamate receptors and the mammalian target of rapamycin (mTOR) signaling pathway in activity-dependent dendritic protein synthesis in hippocampal neurons

Local protein synthesis in neuronal dendrites is critical for synaptic plasticity. However, the signaling cascades that couple synaptic activation to dendritic protein synthesis remain elusive. The purpose of this study is to determine the role of glutamate receptors and the mammalian target of rapamycin (mTOR) signaling in regulating dendritic protein synthesis in live neurons. We first characterized the involvement of various subtypes of glutamate receptors and the mTOR kinase in regulating dendritic synthesis of a green fluorescent protein (GFP) reporter controlled by alphaCaMKII 5' and 3' untranslated regions in cultured hippocampal neurons. Specific antagonists of N-methyl-d-aspartic acid (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and metabotropic glutamate receptors abolished glutamate-induced dendritic GFP synthesis, whereas agonists of NMDA and metabotropic but not AMPA glutamate receptors activated GFP synthesis in dendrites. Inhibitions of the mTOR signaling, as well as its upstream activators, phosphatidylinositol 3-kinase and AKT, blocked NMDA receptor-dependent dendritic GFP synthesis. Conversely, activation of mTOR signaling stimulated dendritic GFP synthesis. In addition, we also found that inhibition of the mTOR kinase blocked dendritic synthesis of the endogenous alphaCaMKII and MAP2 proteins induced by tetanic stimulations in hippocampal slices. These results identify critical roles of NMDA receptors and the mTOR signaling pathway for control of synaptic activity-induced dendritic protein synthesis in hippocampal neurons.     

Leptin reverses long-term potentiation at hippocampal CA1 synapses

The hormone leptin crosses the blood brain barrier and regulates numerous neuronal functions, including hippocampal synaptic plasticity. Here we show that application of leptin resulted in the reversal of long-term potentiation (LTP) at hippocampal CA1 synapses. The ability of leptin to depotentiate CA1 synapses was concentration-dependent and it displayed a distinct temporal profile. Leptin-induced depotentiation was not associated with any change in the paired pulse facilitation ratio or the coefficient of variance, indicating a post-synaptic locus of expression. Moreover, the synaptic activation of NMDA receptors was required for leptin-induced depotentiation as the effects of leptin were blocked by the competitive NMDA receptor antagonist, D-aminophosphovaleric acid (D-AP5). The signaling mechanisms underlying leptin-induced depotentiation involved activation of the calcium/calmodulin-dependent protein phosphatase, calcineurin, but were independent of c- jun NH₂ terminal kinase. Furthermore, leptin-induced depotentiation was accompanied by a reduction in α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor rectification indicating that loss of glutamate receptor 2 (GluR2)-lacking AMPA receptors underlies this process. These data indicate that leptin reverses hippocampal LTP via a process involving calcineurin-dependent internalization of GluR2-lacking AMPA receptors which further highlights the key role for this hormone in regulating hippocampal synaptic plasticity and neuronal development.     

Activation of D1 dopamine receptors increases surface expression of AMPA receptors and facilitates their synaptic incorporation in cultured hippocampal neurons

Considerable evidence indicates that neuroadaptations leading to addiction involve the same cellular processes that enable learning and memory, such as long-term potentiation (LTP), and that psychostimulants influence LTP through dopamine (DA)-dependent mechanisms. In hippocampal CA1 pyramidal neurons, LTP involves insertion of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors into excitatory synapses. We used dissociated cultures to test the hypothesis that D1 family DA receptors influence synaptic plasticity in hippocampal neurons by modulating AMPA receptor trafficking. Brief exposure (5 min) to a D1 agonist increased surface expression of glutamate receptor (GluR)1-containing AMPA receptors by increasing their rate of externalization at extrasynaptic sites. This required the secretory pathway but not protein synthesis, and was mediated mainly by protein kinase A (PKA) with a smaller contribution from Ca2+-calmodulin-dependent protein kinase II (CaMKII). Prior D1 receptor stimulation facilitated synaptic insertion of GluR1 in response to subsequent stimulation of synaptic NMDA receptors with glycine. Our results support a model for synaptic GluR1 incorporation in which PKA is required for initial insertion into the extrasynaptic membrane whereas CaMKII mediates translocation into the synapse. By increasing the size of the extrasynaptic GluR1 pool, D1 receptors may promote LTP. Psychostimulants may usurp this mechanism, leading to inappropriate plasticity that contributes to addiction-related behaviors.     

Essential role for integrin linked kinase in Akt-mediated integrin survival signaling in hippocampal neurons

Activation of integrin receptors in neurons can promote cell survival and synaptic plasticity, but the underlying signal transduction pathway(s) is unknown. We report that integrin signaling prevents apoptosis of embryonic hippocampal neurons by a mechanism involving integrin-linked kinase (ILK) that activates Akt kinase. Activation of integrins using a peptide containing the amino acid sequence EIKLLIS derived from the alpha chain of laminin protected hippocampal neurons from apoptosis induced by glutamate or staurosporine, and increased Akt activity in a beta1 integrin-dependent manner. Transfection of neurons with a plasmid encoding dominant negative Akt blocked the protective effect of the integrin-activating peptide, as did a chemical inhibitor of Akt. Although inhibitors of phosphoinositide-3 (PI3) kinase blocked the protective effect of the peptide, we found no increase in PI3 kinase activity following integrin stimulation suggesting that PI3 kinase was necessary for Akt activity but was not sufficient for the increase in Akt activity following integrin activation. Instead, we show a requirement for ILK in integrin receptor-induced Akt activation. ILK was activated following integrin stimulation and dominant negative ILK blocked integrin-mediated Akt activation and cell survival. Activation of ILK and Akt were also required for neuroprotection by substrate-associated laminin. These results establish a novel pathway that signals cell survival in neurons in response to integrin receptor activation.     

N-methyl-D-aspartate-induced alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA) receptor down-regulation involves interaction of the carboxyl terminus of GluR2/3 with Pick1. Ligand-binding studies using Sindbis vectors carrying AMPA receptor decoys

The dynamics of alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA)-type glutamate receptors, as represented by their exocytosis, endocytosis and cytoskeletal linkage, has often been implicated in N-methyl-d-aspartate (NMDA)-dependent synaptic plasticity. To explore the molecular mechanisms underlying the AMPA receptor dynamics, cultured hippocampal neurons were stimulated with 100 microm NMDA, and the biochemical and pharmacological changes in the ligand binding activity of AMPA receptor complexes and its subunits, GluR1 and GluR2/3, were investigated. The NMDA treatment reduced the total amount of bound [(3)H]AMPA on the surface of the neurons but not in their total membrane fraction. This process was mimicked by a protein kinase C activator, phorbol ester, but blocked by an inhibitor of the same kinase, calphostin C. The NMDA-induced down-regulation of the ligand binding activity was also reflected by the decreased AMPA-triggered channel activity as well as by the cells' reduced immunoreactivity for GluR1. In parallel, the NMDA treatment markedly altered the interaction between the AMPA receptor subunits and their associating molecule(s); the association of PDZ molecules, including Pick1, with GluR2/3 was enhanced in a protein-kinase-C-dependent manner. Viral expression vectors carrying GluR1 and GluR2 C-terminal decoys, both fused to enhanced green fluorescent protein, were transfected into hippocampal neurons to disrupt their interactions. The overexpression of the C-terminal decoy for GluR2 specifically and significantly blocked the NMDA-triggered reduction in [(3)H]AMPA binding, whereas that for GluR1 had no effects. Co-immunoprecipitation using anti-Pick1 antibodies revealed that the overexpressed GluR2 C-terminal decoy indeed prevented Pick1 from interacting with the endogenous GluR2/3. Therefore, these observations suggest that the NMDA-induced down-regulation of the functional AMPA receptors involves the interaction between GluR2/3 subunits and Pick1.     

Role for A kinase-anchoring proteins (AKAPS) in glutamate receptor trafficking and long term synaptic depression

Expression of N-methyl d-aspartate (NMDA) receptor-dependent homosynaptic long term depression at synapses in the hippocampus and neocortex requires the persistent dephosphorylation of postsynaptic protein kinase A substrates. An attractive mechanism for expression of long term depression is the loss of surface AMPA (alpha-amino-3-hydroxy-5-methylisoxazale-4-propionate) receptors at synapses. Here we show that a threshold level of NMDA receptor activation must be exceeded to trigger a stable loss of AMPA receptors from the surface of cultured hippocampal neurons. NMDA also causes displacement of protein kinase A from the synapse, and inhibiting protein kinase A (PKA) activity mimics the NMDA-induced loss of surface AMPA receptors. PKA is targeted to the synapse by an interaction with the A kinase-anchoring protein, AKAP79/150. Disruption of the PKA-AKAP interaction is sufficient to cause a long-lasting reduction in synaptic AMPA receptors in cultured neurons. In addition, we demonstrate in hippocampal slices that displacement of PKA from AKADs occludes synaptically induced long term depression. These data indicate that synaptic anchoring of PKA through association with AKAPs plays an important role in the regulation of AMPA receptor surface expression and synaptic plasticity.     

AMPA receptor-dependent clustering of synaptic NMDA receptors is mediated by Stargazin and NR2A/B in spinal neurons and hippocampal interneurons

Under standard conditions, cultured ventral spinal neurons cluster AMPA- but not NMDA-type glutamate receptors at excitatory synapses on their dendritic shafts in spite of abundant expression of the ubiquitous NMDA receptor subunit NR1. We demonstrate here that the NMDA receptor subunits NR2A and NR2B are not routinely expressed in cultured spinal neurons and that transfection with NR2A or NR2B reconstitutes the synaptic targeting of NMDA receptors and confers on exogenous application of the immediate early gene product Narp the ability to cluster both AMPA and NMDA receptors. The use of dominant-negative mutants of GluR2 further showed that the synaptic targeting of NMDA receptors is dependent on the presence of synaptic AMPA receptors and that synaptic AMPA and NMDA receptors are linked by Stargazin and a MAGUK protein. This system of AMPA receptor-dependent synaptic NMDA receptor localization was preserved in hippocampal interneurons but reversed in hippocampal pyramidal neurons.     

Thymidine phosphorylase and 2-deoxyribose stimulate human endothelial cell migration by specific activation of the integrins alpha 5 beta 1 and alpha V beta 3

Thymidine phosphorylase is an angiogenic factor that is frequently overexpressed in solid tumors, in rheumatoid arthritis, and in response to inflammatory cytokines. Our previous studies showed that cells expressing thymidine phosphorylase stimulated endothelial cell migration in vitro. This was a consequence of the intracellular metabolism of thymidine by thymidine phosphorylase and subsequent extracellular release of 2-deoxyribose. The mechanisms by which 2-deoxyribose might mediate thymidine phosphorylase-induced cell migration in vitro, however, are obscure. Here we show that both thymidine phosphorylase and 2-deoxyribose stimulated the formation of focal adhesions and the tyrosine 397 phosphorylation of focal adhesion kinase in human umbilical vein endothelial cells. Although similar actions occurred upon treatment with the angiogenic factor vascular endothelial growth factor (VEGF), thymidine phosphorylase differed from VEGF in that its effect on endothelial cell migration was blocked by antibodies to either integrin alpha 5 beta 1 or alpha v beta 3, whereas VEGF-induced endothelial cell migration was only blocked by the alpha v beta 3 antibody. Further, thymidine phosphorylase and 2-deoxyribose, but not VEGF, increased the association of both focal adhesion kinase and the focal adhesion-associated protein vinculin with integrin alpha 5 beta 1 and, in intact cells, increased the co-localization of focal adhesion kinase with alpha 5 beta 1. Thymidine phosphorylase and 2-deoxyribose-induced focal adhesion kinase phosphorylation was blocked by the antibodies to alpha 5 beta 1 and alpha v beta 3, directly linking the migration and signaling components of thymidine phosphorylase and 2-deoxyribose action. Cell surface expression of alpha 5 beta 1 was also increased by thymidine phosphorylase and 2-deoxyribose. These experiments are the first to demonstrate a direct effect of thymidine phosphorylase and 2-deoxyribose on signaling pathways associated with endothelial cell migration.     

Activation of D1/D5 dopamine receptors protects neurons from synapse dysfunction induced by amyloid-beta oligomers

Soluble oligomers of the amyloid-β peptide (AβOs) accumulate in the brains of Alzheimer disease (AD) patients and are implicated in synapse failure and early memory loss in AD. AβOs have been shown to impact synapse function by inhibiting long term potentiation, facilitating the induction of long term depression and inducing internalization of both AMPA and NMDA glutamate receptors, critical players in plasticity mechanisms. Because activation of dopamine D1/D5 receptors plays important roles in memory circuits by increasing the insertion of AMPA and NMDA receptors at synapses, we hypothesized that selective activation of D1/D5 receptors could protect synapses from the deleterious action of AβOs. We show that SKF81297, a selective D1/D5 receptor agonist, prevented the reduction in surface levels of AMPA and NMDA receptors induced by AβOs in hippocampal neurons in culture. Protection by SKF81297 was abrogated by the specific D1/D5 antagonist, SCH23390. Levels of AMPA receptor subunit GluR1 phosphorylated at Ser(845), which regulates AMPA receptor association with the plasma membrane, were reduced in a calcineurin-dependent manner in the presence of AβOs, and treatment with SKF81297 prevented this reduction. Establishing the functional relevance of these findings, SKF81297 blocked the impairment of long term potentiation induced by AβOs in hippocampal slices. Results suggest that D1/D5 receptors may be relevant targets for development of novel pharmacological approaches to prevent synapse failure in AD.     

alpha- and betaCaMKII. Inverse regulation by neuronal activity and opposing effects on synaptic strength

We show that alpha and betaCaMKII are inversely regulated by activity in hippocampal neurons in culture: the alpha/beta ratio shifts toward alpha during increased activity and beta during decreased activity. The swing in ratio is approximately 5-fold and may help tune the CaMKII holoenzyme to changing intensities of Ca(2+) signaling. The regulation of CaMKII levels uses distinguishable pathways, one responsive to NMDA receptor blockade that controls alphaCaMKII alone, the other responsive to AMPA receptor blockade and involving betaCaMKII and possibly further downstream effects of betaCaMKII on alphaCaMKII. Overexpression of alphaCaMKII or betaCaMKII resulted in opposing effects on unitary synaptic strength as well as mEPSC frequency that could account in part for activity-dependent effects observed with chronic blockade of AMPA receptors. Regulation of CaMKII subunit composition may be important for both activity-dependent synaptic homeostasis and plasticity.     

Activation of synaptic NMDA receptors induces membrane insertion of new AMPA receptors and LTP in cultured hippocampal neurons

Long-term potentiation (LTP) of excitatory transmission in the hippocampus likely contributes to learning and memory. The mechanisms underlying LTP at these synapses are not well understood, although phosphorylation and redistribution of AMPA receptors may be responsible for this form of synaptic plasticity. We show here that miniature excitatory postsynaptic currents (mEPSCs) in cultured hippocampal neurons reliably demonstrate LTP when postsynaptic NMDA receptors are briefly stimulated with glycine. LTP of these synapses is accompanied by a rapid insertion of native AMPA receptors and by increased clustering of AMPA receptors at the surface of dendritic membranes. Both LTP and glycine-facilitated AMPA receptor insertion are blocked by intracellular tetanus toxin (TeTx), providing evidence that AMPA receptors are inserted into excitatory synapses via a SNARE-dependent exocytosis during LTP.     

Regulation of dendritic excitability by activity-dependent trafficking of the A-type K+ channel subunit Kv4.2 in hippocampal neurons

Voltage-gated A-type K+ channel Kv4.2 subunits are highly expressed in the dendrites of hippocampal CA1 neurons. However, little is known about the subcellular distribution and trafficking of Kv4.2-containing channels. Here we provide evidence for activity-dependent trafficking of Kv4.2 in hippocampal spines and dendrites. Live imaging and electrophysiological recordings showed that Kv4.2 internalization is induced rapidly upon glutamate receptor stimulation. Kv4.2 internalization was clathrin mediated and required NMDA receptor activation and Ca2+ influx. In dissociated hippocampal neurons, mEPSC amplitude depended on functional Kv4.2 expression level and was enhanced by stimuli that induced Kv4.2 internalization. Long-term potentiation (LTP) induced by brief glycine application resulted in synaptic insertion of GluR1-containing AMPA receptors along with Kv4.2 internalization. We also found evidence of Kv4.2 internalization upon synaptically evoked LTP in CA1 neurons of hippocampal slice cultures. These results present an additional mechanism for synaptic integration and plasticity through the activity-dependent regulation of Kv4.2 channel surface expression.     

Activity-dependent regulation of synaptic AMPA receptor composition and abundance by beta3 integrins

At synapses, cell adhesion molecules (CAMs) provide the molecular framework for coordinating signaling events across the synaptic cleft. Among synaptic CAMs, the integrins, receptors for extracellular matrix proteins and counterreceptors on adjacent cells, are implicated in synapse maturation and plasticity and memory formation. However, little is known about the molecular mechanisms of integrin action at central synapses. Here, we report that postsynaptic beta3 integrins control synaptic strength by regulating AMPA receptors (AMPARs) in a subunit-specific manner. Pharmacological perturbation targeting beta3 integrins promotes endocytosis of GluR2-containing AMPARs via Rap1 signaling, and expression of beta3 integrins produces robust changes in the abundance and composition of synaptic AMPARs without affecting dendritic spine structure. Importantly, homeostatic synaptic scaling induced by activity deprivation elevates surface expression of beta3 integrins, and in turn, beta3 integrins are required for synaptic scaling. Our findings demonstrate a key role for integrins in the feedback regulation of excitatory synaptic strength.     

Expression mechanisms underlying long-term potentiation: a postsynaptic view

This review summarizes the various experiments that have been carried out to determine if the expression of long-term potentiation (LTP), in particular N-methyl-D-aspartate (NMDA) receptor-dependent LTP, is presynaptic or postsynaptic. Evidence for a presynaptic expression mechanism comes primarily from experiments reporting that glutamate overflow is increased during LTP and from experiments showing that the failure rate decreases during LTP. However, other experimental approaches, such as monitoring synaptic glutamate release by recording astrocytic glutamate transporter currents, have failed to detect any change in glutamate release during LTP. In addition, the discovery of silent synapses, in which LTP rapidly switches on alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor function at NMDA-receptor-only synapses, provides a postsynaptic mechanism for the decrease in failures during LTP. It is argued that the preponderance of evidence favours a postsynaptic expression mechanism, whereby NMDA receptor activation results in the rapid recruitment of AMPA receptors as well as a covalent modification of synaptic AMPA receptors.     

alpha1,3 fucosyltransferase-VII up-regulates the mRNA of alpha5 integrin and its biological function

After transfection of alpha1,3fucosyltransferase (FucT)-VII cDNA into H7721 human hepatocarcinoma cells, the expression of alpha5, but not beta1 integrin was significantly up-regulated. This was evidenced by the increase of alpha5 integrin on cell surface as well as the increase of alpha5 mRNA and protein in the cells. However, the expressions of sialyl Lewis X (SLe(x), the product of alpha1,3FucT-VII) on both alpha5 and beta1 integrin subunits were unchanged. Concomitantly, the tyrosine autophosphorylated FAK and dephosphorylated Src (FAK and Src involve in the signal transduction of integrin alpha5beta1) were up-regulated, while the Tyr-527 phosphorylated Src was down-regulated. The above-mentioned alterations were correlated to the expressions of alpha1,3FucT-VII in different alpha1,3FucT-VII transfected H7721 cell lines. In addition, after alpha1,3FucT-VII transfection, cell adhesion to fibronectin (Fn) and chemotaxic cell migration were obviously promoted. The cell adhesion could be blocked by alpha5 integrin antibody, and cell migration was obviously attenuated by the antibodies to both alpha5 integrin and SLe(x). These findings suggest that the increased surface alpha5 integrin caused by the up-regulation of alpha5 mRNA promotes the cell adhesion to Fn, cell migratiom, and Fn-induced signaling of alpha5beta1 integrin. The up-regulation of surface SLe(x) originated from the over expression of alpha1,3FucT-VII also led to the stimulation of cell migration. This is the first time to report that alpha1,3FucT-VII can regulate the mRNA expression of integrin.     

Selective cholinergic depletion in medial septum leads to impaired long term potentiation and glutamatergic synaptic currents in the hippocampus

Cholinergic depletion in the medial septum (MS) is associated with impaired hippocampal-dependent learning and memory. Here we investigated whether long term potentiation (LTP) and synaptic currents, mediated by alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA) and N-methyl-D-aspartate (NMDA) receptors in the CA1 hippocampal region, are affected following cholinergic lesions of the MS. Stereotaxic intra-medioseptal infusions of a selective immunotoxin, 192-saporin, against cholinergic neurons or sterile saline were made in adult rats. Four days after infusions, hippocampal slices were made and LTP, whole cell, and single channel (AMPA or NMDA receptor) currents were recorded. Results demonstrated impairment in the induction and expression of LTP in lesioned rats. Lesioned rats also showed decreases in synaptic currents from CA1 pyramidal cells and synaptosomal single channels of AMPA and NMDA receptors. Our results suggest that MS cholinergic afferents modulate LTP and glutamatergic currents in the CA1 region of the hippocampus, providing a potential synaptic mechanism for the learning and memory deficits observed in the rodent model of selective MS cholinergic lesioning.     

Beta1 integrin/focal adhesion kinase-mediated signaling induces intercellular adhesion molecule 1 and receptor activator of nuclear factor kappaB ligand on osteoblasts and osteoclast maturation

We have assessed characteristics of primary human osteoblasts, shedding light on signaling mediated by beta1 integrin. beta1 integrins are major receptors for these matrix glycoproteins. 1) Integrins beta1, alpha2, alpha3, alpha4, alpha5, alpha6, and alphav were highly expressed on primary osteoblasts. 2) Engagement of beta1 integrins on osteoblasts by cross-linking with specific antibody or ligand matrices, such as fibronectin or collagen, augmented expression of intercellular adhesion molecule 1 (ICAM-1) and receptor activator of nuclear factor kappaB ligand (RANKL) on the surface. 3) Up-regulation of ICAM-1 and RANKL on osteoblasts by beta1 stimulation was completely abrogated by pretreatment with herbimycin A and genistein, tyrosine kinase inhibitors, or transfection of dominant negative truncations of focal adhesion kinase (FAK). 4) Engagement of beta1 integrins on osteoblasts induced tartrate-resistant acid phosphatase-positive multinuclear cell formation in the coculture system of osteoblasts and peripheral monocytes. 5) Up-regulation of tartrate-resistant acid phosphatase-positive multinuclear cell formation by beta1 stimulation was completely abrogated by transfection of dominant negative truncations of FAK. Our results indicate that beta1 integrin-dependent adhesion of osteoblasts to bone matrices induces ICAM-1 and RANKL expression and osteoclast formation via tyrosine kinase, especially FAK. We here propose that beta1 integrin/FAK-mediated signaling on osteoblasts could be involved in ICAM-1- and RANKL-dependent osteoclast maturation.