Relevance of N-terminal residues for amyloid-β binding to platelet integrin αIIbβ3, integrin outside-in signaling and amyloid-β fibril formation

A pathological hallmark of Alzheimer's disease (AD) is the aggregation of amyloid-β peptides (Aβ) into fibrils, leading to deposits in cerebral parenchyma and vessels known as cerebral amyloid angiopathy (CAA). Platelets are major players of hemostasis but are also implicated in AD. Recently we provided strong evidence for a direct contribution of platelets to AD pathology. We found that monomeric Aβ40 binds through its RHDS sequence to integrin α_IIbβ₃, and promotes the formation of fibrillar Aβ aggregates by the secretion of adenosine diphosphate (ADP) and the chaperone protein clusterin (CLU) from platelets. Here we investigated the molecular mechanisms of Aβ binding to integrin α_IIbβ₃ by using Aβ11 and Aβ16 peptides. These peptides include the RHDS binding motif important for integrin binding but lack the central hydrophobic core and the C-terminal sequence of Aβ. We observed platelet adhesion to truncated N-terminal Aβ11 and Aβ16 peptides that was not mediated by integrin α_IIbβ₃. Thus, no integrin outside-in signaling and reduced CLU release was detected. Accordingly, platelet mediated Aβ fibril formation was not observed. Taken together, the RHDS motif of Aβ is not sufficient for Aβ binding to platelet integrin α_IIbβ₃ and platelet mediated Aβ fibril formation but requires other recognition or binding motifs important for platelet mediated processes in CAA. Thus, increased understanding of the molecular mechanisms of Aβ binding to platelet integrin α_IIbβ₃ is important to understand the role of platelets in amyloid pathology.     

Kindlin-3 mediates integrin αLβ2 outside-in signaling, and it interacts with scaffold protein receptor for activated-C kinase 1 (RACK1)

Integrins are heterodimeric type I membrane cell adhesion molecules that are involved in many biological processes. Integrins are bidirectional signal transducers because their cytoplasmic tails are docking sites for cytoskeletal and signaling molecules. Kindlins are cytoplasmic molecules that mediate inside-out signaling and activation of the integrins. The three kindlin paralogs in humans are kindlin-1, -2, and -3. Each of these contains a 4.1-ezrin-radixin-moesin (FERM) domain and a pleckstrin homology domain. Kindlin-3 is expressed in platelets, hematopoietic cells, and endothelial cells. Here we show that kindlin-3 is involved in integrin αLβ2 outside-in signaling. It also promotes micro-clustering of integrin αLβ2. We provide evidence that kindlin-3 interacts with the receptor for activated-C kinase 1 (RACK1), a scaffold protein that folds into a seven-blade propeller. This interaction involves the pleckstrin homology domain of kindlin-3 and blades 5-7 of RACK1. Using the SKW3 human T lymphoma cells, we show that integrin αLβ2 engagement by its ligand ICAM-1 promotes the association of kindlin-3 with RACK1. We also show that kindlin-3 co-localizes with RACK1 in polarized SKW3 cells and human T lymphoblasts. Our findings suggest that kindlin-3 plays an important role in integrin αLβ2 outside-in signaling.     

Vinculin activates inside-out signaling of integrin αIIbβ3 in Chinese hamster ovary cells

Although vinculin is used frequently as a marker for integrin-mediated focal adhesion complexes, how it regulates the activation of integrin is mostly unknown. In this study, we examined whether vinculin would activate integrin in Chinese hamster ovary (CHO) cells expressing human integrin αIIbβ3. Silencing of vinculin by lentiviral transduction with a short hairpin RNA sequence affected the binding of PAC-1 (an antibody recognizing activated human αIIbβ3) to a constitutively active form of αIIbβ3 (α6Bβ3) expressed on CHO cells, while its inhibitory effects were much weaker than those of talin-1. Overexpression of an active form of vinculin without intramolecular interactions, but not the full length one, induced PAC-1 binding to native αIIbβ3 expressed on CHO cells in a manner dependent on talin-1. On the other hand, silencing of talin-1, but not vinculin, failed to induce cell spreading of α6Bβ3-CHO cells on fibrinogen, even in the presence of PT 25-2, a monoclonal antibody that activates αIIbβ3. Thus, an active form of vinculin could induce αIIbβ3 inside-out signaling through the actions of talin-1, while vinculin was dispensable for outside-in signaling.     

G(12/13) signaling pathways substitute for integrin αIIbβ3-signaling for thromboxane generation in platelets

Background

We have previously shown that ADP-induced TXA₂ generation requires signaling from αIIbβ3 integrin in platelets. Here we observed that, unlike ADP, protease-activated receptor (PAR)-mediated TXA₂ generation occurs independently of αIIbβ3. PAR agonists, but not ADP, activate G_12/13 signaling pathways. Hence, we evaluated the role of these pathways in TXA₂ generation.

Principal Findings

Inhibition of ADP-induced thromboxane generation by fibrinogen receptor antagonist SC57101 was rescued by co-stimulation of G_12/13 pathways with YFLLRNP. This observation suggested an existence of a common signaling effector downstream of integrins and G_12/13 pathways. Hence, we evaluated role of three potential tyrosine kinases; c-Src, Syk and FAK (Focal Adhesion Kinase) that are known to be activated by integrins. c-Src and Syk kinase did not play a role in ADP-induced functional responses in platelets. Selective activation of G_12/13 pathways resulted in the activation of FAK, in the absence of integrin signaling. Interestingly, αIIbβ3-mediated FAK activation occurred in a Src family kinase (SFK)-independent manner whereas G_12/13 pathway caused FAK activation in a SFK and RhoA-dependent manner. A FAK selective inhibitor TAE-226, blocked TXA₂ generation. However, in comparison to WT mice, Pf4-Cre/Fak-Floxed mice did not show any difference in platelet TXA₂ generation.

Conclusions

Therefore, we conclude that differential activation of FAK occurs downstream of Integrins and G_12/13 pathways. However, the common effector molecule, possibly a tyrosine kinase downstream of integrins and G_12/13 pathways contributing to TXA₂ generation in platelets remains elusive.     

Serine/threonine phosphatases regulate platelet αIIbβ3 integrin receptor outside-in signaling mechanisms and clot retraction

Aims

We studied the role of serine/threonine phosphatases (PSTPs) on α_IIbβ₃ signaling and the potential selectivity of the level of PSTP inhibition with okadaic acid (OA) on α_IIbβ₃ signaling for regulation of platelet aggregation and clot retraction.

Main methods

We used washed platelets from normal donors and OA as inhibitor of PSTPs. Clot retraction was induced by 1 U/mL of thrombin. Reorganized cytoskeleton was isolated from Triton X-100 lysed platelets. The presence of proteins incorporated to the cytoskeleton was assayed by immunoblotting with specific antibodies.

Key findings

We found that both 100 and 500 nM OA blocked platelet mediated clot retraction. In contrast, only 500 nM OA inhibited thrombin-induced inside-out α_IIbβ₃ activation, platelet aggregation, and cytoskeletal reorganization. Among markers of α_IIbβ₃ outside-in signaling, 500 nM OA inhibited the incorporation to the cytoskeleton of syk, src, and FAK (Focal Adhesion Kinase) tyrosine kinases and the incorporation and phosphorylation at Tyr⁷⁵⁹ of the β₃ chain of α_IIbβ₃, while 100 nM OA only inhibited the FAK translocation and its tyrosine phosphorylation.

Significance

The level of inhibition of PSTPs by low or high OA concentration (33% and 73% inhibition, respectively) in intact whole cells differentially regulates platelet aggregation and integrin signaling, but have a common effect in blocking clot retraction. The latter may be associated with the presence of phosphorylated FAK in the cytoskeleton. This study reveals a novel target for anti-platelet treatment to block clot retraction without affecting the platelet hemostatic function by a partial inhibition of PSTPs.     

Functional interaction of CD154 protein with α5β1 integrin is totally independent from its binding to αIIbβ3 integrin and CD40 molecules

In addition to its classical CD40 receptor, CD154 also binds to αIIbβ3, α5β1, and αMβ2 integrins. Binding of CD154 to these receptors seems to play a key role in the pathogenic processes of chronic inflammation. This investigation was aimed at analyzing the functional interaction of CD154 with CD40, αIIbβ3, and α5β1 receptors. We found that the binding affinity of CD154 for αIIbβ3 is ～4-fold higher than for α5β1. We also describe the generation of sCD154 mutants that lost their ability to bind CD40 or αIIbβ3 and show that CD154 residues involved in its binding to CD40 or αIIbβ3 are distinct from those implicated in its interaction to α5β1, suggesting that sCD154 may bind simultaneously to different receptors. Indeed, sCD154 can bind simultaneously to CD40 and α5β1 and biologically activate human monocytic U937 cells expressing both receptors. The simultaneous engagement of CD40 and α5β1 activates the mitogen-activated protein kinases, p38, and extracellular signal-related kinases 1/2 and synergizes in the release of inflammatory mediators MMP-2 and -9, suggesting a cross-talk between these receptors.     

Regulation and roles of PI3Kβ, a major actor in platelet signaling and functions

Phosphoinositide 3-kinases (PI3Ks) are important signaling enzymes involved in the regulation of a number of critical cell functions. Significant progress has been made during the last few years in defining the implication of individual PI3K isoforms. The role of the class IA PI3Kβ in different cell types has only been recently uncovered by the use of isoform-selective inhibitors and the development of mouse models harboring p110β catalytic subunit knock-out or germline knock-in of a kinase-dead allele of p110β. Although it is classically admitted that class IA PI3Ks are activated by receptor tyrosine kinases through recruitment of the regulatory subunits to specific tyrosine phosphorylated motifs via their SH2 domains, PI3Kβ is activated downstream of G protein-coupled receptors, and by co-operation between heterotrimeric G proteins and tyrosine kinases. PI3Kβ has been extensively studied in platelets where it appears to play an important role downstream of ITAM signaling, G protein-coupled receptors and aIIbβ3 integrin. Accordingly, mouse exhibiting p110β inactivation selectively in megakaryocyte/platelets are resistant to thromboembolism induced by carotid injury. The present review summarizes recent data concerning the mechanisms of PI3Kβ regulation and the roles of this PI3K isoform in blood platelet functions and other cell types.     

Regulation of integrin αIIbβ3 ligand binding and signaling by the metal ion binding sites in the β I domain

The ability of αIIbβ3 to bind ligands and undergo outside-in signaling is regulated by three divalent cation binding sites in the β I domain. Specifically, the metal ion-dependent adhesion site (MIDAS) and the synergistic metal binding site (SyMBS) are thought to be required for ligand binding due to their synergy between Ca(2+) and Mg(2+). The adjacent to MIDAS (ADMIDAS) is an important ligand binding regulatory site that also acts as a critical link between the β I and hybrid domains for signaling. Mutations in this site have provided conflicting results for ligand binding and adhesion in different integrins. We have mutated the β3 SyMBS and ADMIDAS. The SyMBS mutant abolished ligand binding and outside-in signaling, but when an activating glycosylation mutation in the αIIb Calf 2 domain was introduced, the ligand binding affinity and signaling were restored. Thus, the SyMBS is important but not absolutely required for integrin bidirectional signaling. The ADMIDAS mutants showed reduced ligand binding affinity and abolished outside-in signaling, and the activating glycosylation mutation could fully restore integrin signaling of the ADMIDAS mutant. We propose that the ADMIDAS ion stabilizes the low-affinity state when the integrin headpiece is in the closed conformation, whereas it stabilizes the high-affinity state when the headpiece is in the open conformation with the swung-out hybrid domain.     

Kindlins, integrin activation and the regulation of talin recruitment to αIIbβ3

Talins and kindlins bind to the integrin β3 cytoplasmic tail and both are required for effective activation of integrin αIIbβ3 and resulting high-affinity ligand binding in platelets. However, binding of the talin head domain alone to β3 is sufficient to activate purified integrin αIIbβ3 in vitro. Since talin is localized to the cytoplasm of unstimulated platelets, its re-localization to the plasma membrane and to the integrin is required for activation. Here we explored the mechanism whereby kindlins function as integrin co-activators. To test whether kindlins regulate talin recruitment to plasma membranes and to αIIbβ3, full-length talin and kindlin recruitment to β3 was studied using a reconstructed CHO cell model system that recapitulates agonist-induced αIIbβ3 activation. Over-expression of kindlin-2, the endogenous kindlin isoform in CHO cells, promoted PAR1-mediated and talin-dependent ligand binding. In contrast, shRNA knockdown of kindlin-2 inhibited ligand binding. However, depletion of kindlin-2 by shRNA did not affect talin recruitment to the plasma membrane, as assessed by sub-cellular fractionation, and neither over-expression of kindlins nor depletion of kindlin-2 affected talin interaction with αIIbβ3 in living cells, as monitored by bimolecular fluorescence complementation. Furthermore, talin failed to promote kindlin-2 association with αIIbβ3 in CHO cells. In addition, purified talin and kindlin-3, the kindlin isoform expressed in platelets, failed to promote each other's binding to the β3 cytoplasmic tail in vitro. Thus, kindlins do not promote initial talin recruitment to αIIbβ3, suggesting that they co-activate integrin through a mechanism independent of recruitment.     

A conserved lipid-binding loop in the kindlin FERM F1 domain is required for kindlin-mediated αIIbβ3 integrin coactivation

The activation of heterodimeric integrin adhesion receptors from low to high affinity states occurs in response to intracellular signals that act on the short cytoplasmic tails of integrin β subunits. Binding of the talin FERM (four-point-one, ezrin, radixin, moesin) domain to the integrin β tail provides one key activation signal, but recent data indicate that the kindlin family of FERM domain proteins also play a central role. Kindlins directly bind integrin β subunit cytoplasmic domains at a site distinct from the talin-binding site, and target to focal adhesions in adherent cells. However, the mechanisms by which kindlins impact integrin activation remain largely unknown. A notable feature of kindlins is their similarity to the integrin-binding and activating talin FERM domain. Drawing on this similarity, here we report the identification of an unstructured insert in the kindlin F1 FERM domain, and provide evidence that a highly conserved polylysine motif in this loop supports binding to negatively charged phospholipid head groups. We further show that the F1 loop and its membrane-binding motif are required for kindlin-1 targeting to focal adhesions, and for the cooperation between kindlin-1 and -2 and the talin head in αIIbβ3 integrin activation, but not for kindlin binding to integrin β tails. These studies highlight the structural and functional similarities between kindlins and the talin head and indicate that as for talin, FERM domain interactions with acidic membrane phospholipids as well β-integrin tails contribute to the ability of kindlins to activate integrins.     

Structural and functional aspects of decorsin and its analog as recognized by integrin αIIbβ3

Decorsin is an antagonist of platelet glycoprotein integrin αIIbβ3 on platelets; the protein is 39 amino acids long with three disulfide bridges in its tertiary structure. To demonstrate decorsin’s mechanism of action, we applied the computational virtual technique and platelet aggregation inhibition assay, which showed that the flanking amino-acid residues of the Arg–Gly–Asp (RGD) motif play an important role in platelet aggregation. The computational simulations revealed that the RGD motif mainly contributes to the stability of the complex when decorsion interacts with integrin αIIbβ3. However, the C-terminal residues, such as 34A→W and 35D→R, was also found to possibly play a key role in their binding structures. Moreover, we produced a decorsin analog (A34W plus D35R decorsin), in which the 34A (alanine) and 35D (aspartic acid) residues were respectively substituted by W (tryptophan) and R (arginine). This isoform was then recombinantly expressed in Escherichia coli. Intriguingly, this mutant type showed higher anti-platelet aggregation activity than the wildtype. Our study may further contribute to finding decorsin mutants with higher anti-platelet aggregation activity.     

C-glyco“RGD” as αIIbβ3 and αvβ integrin ligands for imaging applications: Synthesis,in vitro evaluation and molecular modeling

The design of conjugates displaying simultaneously high selectivity and high affinity for different subtypes of integrins is a current challenge. The arginine-glycine-aspartic acid amino acid sequence (RGD) is one of the most efficient short peptides targeting these receptors. We report herein the development of linear and cyclic fluoro-C-glycoside“RGD” conjugates, taking advantage of the robustness and hydrophilicity of C-glycosides. As attested by in vitro evaluation, the design of these C-glyco“RGD” with a flexible three-carbon triazolyl linker allows distinct profiles towards α_IIbβ₃ and α_vβ₃ integrins. Molecular-dynamics simulations confirm the suitability of cyclic C-glyco-c(RGDfC) to target α_vβ₃ integrin. These C-glyco”RGD” could become promising biological tools in particular for Positron Emission Tomography imaging.     

Construction and characterization of a novel chimeric antibody c3C7 specific for the integrin αIIbβ3 complex

A murine monoclonal antibody (mAb) 3C7 against integrin αIIbβ3 was previously obtained as a potential antithrombotic agent in our laboratory. The epitope of 3C7 is a specific conformation of the αIIbβ3 complex, but not either of the two subunits, which makes it different from abciximab, a supplementary antibody drug used in percutaneous coronary intervention which has a cross-reaction with other integrins sharing the β3 subunit. To reduce the human anti-mouse antibody reactions of 3C7, the variable regions of this antibody were cloned and fused with the constant counterparts of human IgG1. Two vectors of light and heavy chains were constructed and co-transfected into CHO-dhfr ^? cells. The chimeric antibody c3C7 was purified and the properties of c3C7 were compared with 3C7. Identical to its parent antibody 3C7, c3C7 binds to the αIIbβ3 complex, but not to either of the subunits. The K _d value of c3C7 was in the same order of magnitude as 3C7 (1.570?±?0.326 vs 0.780?±?0.182 nmol/L). Human platelet aggregation induced by adenosine diphosphate was effectively inhibited by c3C7 in a dose-dependent manner. In conclusion, after the modification, c3C7 retained the properties of its parent mAb with no loss of its biological activity. Therefore, c3C7 has the potential to become a novel agent for the treatment of thrombosis.     

The α7β1 integrin in muscle development and disease

The alpha7beta1 integrin is a laminin receptor on the surface of skeletal myoblasts and myofibers. Alternative forms of both the alpha7 and beta1 chains are expressed in a developmentally regulated fashion during myogenesis. These different alpha7beta1 isoforms localize at specific sites on myofibers and appear to have distinct functions in skeletal muscle. These functions include the migration and proliferation of developing myoblasts, the formation and integrity of neuromuscular and myotendinous junctions, and the "gluing" together of muscle fibers that is essential to the generation of contractile force. The alpha7beta1 integrin appears to be both directly and indirectly causally related to several muscle diseases. Enhanced expression of alpha7beta1-mediated linkage of the extracellular matrix is seen in Duchenne muscular dystrophy and may compensate for the absence of the dystrophin-mediated linkage. Downregulation of expression of the integrin may contribute to the development of pathology in congenital laminin deficiencies. Mutations in the alpha7 integrin gene underlie additional congenital muscle diseases. The functional roles of this integrin in the formation and stability of the neuromuscular and myotendinous junctions and its localization between fibers suggest that altered expression or function of this integrin may have widespread involvement in other myopathies. The localization of the alpha7 gene at human chromosome 12q13 is a useful clue for focusing such studies.     

RAGE and αVβ3-integrin are essential for suPAR signaling in podocytes

The soluble urokinase plasminogen activator receptor (suPAR) has been implicated in the pathogenesis of kidney diseases including primary and recurrent focal and segmental glomerulosclerosis (FSGS), diabetic nephropathy, and acute kidney injuries (AKI). Elevated serum suPAR concentration is a negative prognostic indicator in multiple critical clinical conditions. This study has examined the initial transduction steps used by suPAR in cultured mouse podocytes. We now report that the receptor for advanced glycation end-products (RAGE) co-immunoprecipitates with αV and β3 integrin subunits, which have been previously shown to initiate suPAR signal transduction at the podocyte cell surface. siRNA knock-down of RAGE attenuated Src phosphorylation evoked by either suPAR or by glycated albumin (AGE-BSA), a prototypical RAGE agonist. suPAR effects on Src phosphorylation were also blocked by the structurally dissimilar RAGE antagonists FPS-ZM1 and azeliragon, as well as by cilengitide, an inhibitor of outside-in signaling through αV-integrins. FPS-ZM1 also blocked Src phosphorylation evoked by AGE-BSA. FPS-ZM1 blocked increases in cell surface TRPC6 abundance, cytosolic reactive oxygen species (ROS) and activation of the small GTPase Rac1 evoked by either suPAR or AGE-BSA. In addition, FPS-ZM1 inhibited Src phosphorylation evoked by serum collected from a patient with recurrent FSGS during a relapse. The magnitude of this inhibition was indistinguishable from the effect produced by a neutralizing antibody against suPAR. These data suggest that orally bioavailable small molecule RAGE antagonists could represent a useful therapeutic strategy for a wide range of clinical conditions associated with elevated serum suPAR, including primary FSGS and AKI.     

Galectin-3– and phospho-caveolin-1–dependent outside-in integrin signaling mediates the EGF motogenic response in mammary cancer cells

In murine mammary epithelial cancer cells, galectin-3 binding to β1,6-acetylglucosaminyltransferase V (Mgat5)–modified N-glycans restricts epidermal growth factor (EGF) receptor mobility in the plasma membrane and acts synergistically with phospho-caveolin-1 to promote integrin-dependent matrix remodeling and cell migration. We show that EGF signaling to RhoA is galectin-3 and phospho-caveolin-1 dependent and promotes the formation of transient, actin-rich, circular dorsal ruffles (CDRs), cell migration, and fibronectin fibrillogenesis via Src- and integrin-linked kinase (ILK)–dependent signaling. ILK, Src, and galectin-3 also mediate EGF stimulation of caveolin-1 phosphorylation. Direct activation of integrin with Mn²⁺ induces galectin-3, ILK, and Src-dependent RhoA activation and caveolin-1 phosphorylation. This suggests that in response to EGF, galectin-3 enables outside-in integrin signaling stimulating phospho-caveolin-1–dependent RhoA activation, actin reorganization in CDRs, cell migration, and fibronectin remodeling. Similarly, caveolin-1/galectin-3–dependent EGF signaling induces motility, peripheral actin ruffling, and RhoA activation in MDA-MB-231 human breast carcinoma cells, but not HeLa cells. These studies define a galectin-3/phospho-caveolin-1/RhoA signaling module that mediates integrin signaling downstream of growth factor activation, leading to actin and matrix remodeling and tumor cell migration in metastatic cancer cells.     

Cystine-knot peptides engineered with specificities for α(IIb)β(3) or α(IIb)β(3) and α(v)β(3) integrins are potent inhibitors of platelet aggregation

A truncated form of the Agouti‐related protein (AgRP), a member of the cystine‐knot family, has shown promise as a scaffold for engineering novel peptides with new molecular recognition properties. In this study, we replaced a constrained six amino acid loop in AgRP with a nine amino acid loop containing an Arg–Gly–Asp integrin recognition motif, and randomized the neighboring residues to create a library of ～20 million AgRP variants. We displayed the AgRP mutants as fusions on the surface of yeast and used high‐throughput fluorescence‐activated cell sorting (FACS) to isolate peptides that bound specifically to the platelet integrin α_IIbβ₃, a clinically important target for the prevention and treatment of thrombosis. These AgRP peptides had equilibrium dissociation (K_D) constants for α_IIbβ₃ integrin ranging from 60 to 90 nM, and did not bind to α_vβ₃, α_vβ₅, or α₅β₁ integrins. Using an alternate library screening strategy, we identified AgRP peptides that bound to both α_IIbβ₃ and α_vβ₃ integrins with K_D values ranging from 40 to 70 nM and 20 to 30 nM, respectively, and did not bind to α_vβ₅ or α₅β₁ integrins. Unique consensus sequences were identified within both series of AgRP peptides suggesting alternative molecular recognition events that dictate different integrin binding specificities. In addition, the engineered AgRP peptides prevented platelet aggregation as well as or slightly better than the FDA‐approved cyclic peptide eptifibatide. Collectively, these data demonstrate that cystine‐knot peptides can be generated with high affinity and specificity to closely‐related integrins, and provide insights into molecular interactions between small, structured peptide ligands and their receptors. Copyright © 2010 John Wiley & Sons, Ltd.     

Mechanotransduction activates α₅β₁ integrin and PI3K/Akt signaling pathways in mandibular osteoblasts

It is unclear how bone cells at different sites detect mechanical loading and how site-specific mechanotransduction affects bone homeostasis. To differentiate the anabolic mechanical responses of mandibular cells from those of calvarial and long bone cells, we isolated osteoblasts from C57B6J mouse bones, cultured them for 1week, and subjected them to therapeutic low intensity pulsed ultrasound (LIPUS). While the expression of the marker proteins of osteoblasts and osteocytes such as alkaline phosphatase and FGF23, as well as Wnt1 and β-catenin, was equally upregulated, the expression of mandibular osteoblast messages related to bone remodeling and apoptosis differed from that of messages of other osteoblasts, in that the messages encoding the pro-remodeling protein RANKL and the anti-apoptotic protein Bcl-2 were markedly upregulated from the very low baseline levels. Blockage of the PI3K and α(5)β(1) integrin pathways showed that the mandibular osteoblast required mechanotransduction downstream of α(5)β(1) integrin to upregulate expression of the proteins β-catenin, p-Akt, Bcl-2, and RANKL. Mandibular osteoblasts thus must be mechanically loaded to preserve their capability to promote remodeling and to insure osteoblast survival, both of which maintain intact mandibular bone tissue. In contrast, calvarial Bcl-2 is fully expressed, together with ILK and phosphorylated mTOR, in the absence of LIPUS. The antibody blocking α(5)β(1) integrin suppressed both the baseline expression of all calvarial proteins examined and the LIPUS-induced expression of all mandibular proteins examined. These findings indicate that the cellular environment, in addition to the tridermic origin, determines site-specific bone homeostasis through the remodeling and survival of osteoblastic cells. Differentiated cells of the osteoblastic lineage at different sites transmit signals through transmembrane integrins such as α(5)β(1) integrin in mandibular osteoblasts, whose signaling may play a major role in controlling bone homeostasis.     

Phosphoenolpyruvate-dependent inhibition of collagen biosynthesis, α2β1 integrin and IGF-I receptor signaling in cultured fibroblasts

The mechanism of collagen biosynthesis regulation is not fully understood. The finding that prolidase plays an important role in collagen biosynthesis and phosphoenolpyruvate inhibits prolidase activity "in vitro" led to evaluate its effect on collagen biosynthesis in cultured human skin fibroblasts. Confluent fibroblasts were treated with millimolar concentrations (1-4 mM) of phosphoenolpyruvate monopotassium salt (PEP) for 24 h. It was found that PEP-dependent decrease in prolidase activity and expression was accompanied by parallel decrease in collagen biosynthesis. However, the experiments with inhibitor of PEP production, 3-mercaptopicolinate revealed no direct correlation between collagen biosynthesis and prolidase activity and expression. Since insulin-like growth factor (IGF-I) is the most potent stimulator of both collagen biosynthesis and prolidase activity, and prolidase is regulated by beta(1) integrin signaling, the effect of PEP on IGF-I receptor (IGF-IR) and beta(1) integrin receptor expressions were evaluated. It was found that the exposure of the cells to 4 mM PEP contributed to a decrease in IGF-IR and beta(1) integrin receptor expressions. The data suggest that PEP-dependent decrease of collagen biosynthesis in cultured human skin fibroblasts may undergo through depression of alpha(2)beta(1) integrin and IGF-IR signaling. The hypothetical mechanism of the role of prolidase in IGF-IR, beta(1) integrin receptor expressions, and clinical significance of the process are discussed.