Hisashi Kato

Adiponectin Acts as an Endogenous Antithrombotic Factor

Objective—Obesity is a common risk factor in insulin resistance and cardiovascular diseases. Although hypoadiponectinemia is associated with obesity-related metabolic and vascular diseases, the role of adiponectin in thrombosis remains elusive. Methods and Results—We investigated platelet thrombus formation in adiponectin knockout (APN-KO) male mice (8 to 12 weeks old) fed on a normal diet. There was no significant difference in platelet counts or coagulation parameters between wild-type (WT) and APN-KO mice. However, APN-KO mice showed an accelerated thrombus formation on carotid arterial injury with a He-Ne laser (total thrombus volume: 13.36±4.25×107 arbitrary units for APN-KO and 6.74±2.87×107 arbitrary units for WT; n=10; P<0.01). Adenovirus-mediated supplementation of adiponectin attenuated the enhanced thrombus formation. In vitro thrombus formation on a type I collagen at a shear rate of 250 s−1, as well as platelet aggregation induced by low concentrations of agonists, was enhanced in APN-KO mice, and recombinant adiponectin inhibited the enhanced platelet aggregation. In WT mice, adenovirus-mediated overexpression of adiponectin additionally attenuated thrombus formation. Conclusion—Adiponectin deficiency leads to enhanced thrombus formation and platelet aggregation. The present study reveals a new role of adiponectin as an endogenous antithrombotic factor.

Visualizing extravasation dynamics of metastatic tumor cells.

Little is known about how metastatic cancer cells arrest in small capillaries and traverse the vascular wall during extravasation in vivo. Using real-time intravital imaging of human tumor cells transplanted into transparent zebrafish, we show here that extravasation of cancer cells is a highly dynamic process that involves the modulation of tumor cell adhesion to the endothelium and intravascular cell migration along the luminal surface of the vascular wall. Tumor cells do not damage or induce vascular leak at the site of extravasation, but rather induce local vessel remodeling characterized by clustering of endothelial cells and cell-cell junctions. Intravascular locomotion of tumor cells is independent of the direction of blood flow and requires β1-integrin-mediated adhesion to the blood-vessel wall. Interestingly, the expression of the pro-metastatic gene Twist in tumor cells increases their intravascular migration and extravasation through the vessel wall. However, in this case, Twist expression causes the tumor cells to switch to a β1-integrin-independent mode of extravasation that is associated with the formation of large dynamic rounded membrane protrusions. Our results demonstrate that extravasation of tumor cells is a highly dynamic process influenced by metastatic genes that target adhesion and intravascular migration of tumor cells, and induce endothelial remodeling.

Cyclic GMP and Protein Kinase G Control a Src-Containing Mechanosome in Osteoblasts

Drugs that activate protein kinase G could mimic the bone-building effects of mechanical stimulation. Building Bone The loss of bone density that afflicts individuals with osteoporosis makes them more vulnerable to bone fractures. One way to counteract decreases in bone density is through exercise, which mechanically stimulates bone tissue and initiates proliferation in bone-forming cells. Alternatively, the signaling pathways that mediate this proliferative response could be therapeutically activated to mimic the effects of mechanical stimulation. Nitric oxide (NO) is a second messenger that is produced in response to mechanical stimulation; it triggers production of cyclic GMP (cGMP) and, consequently, activation of protein kinase G (PKG). Rangaswami et al. delineated a pathway in mechanically stimulated osteoblasts whereby activation of PKGII signaling ultimately leads to a proliferative response. Mechanical stimuli triggered the formation of a complex containing PKGII, the tyrosine kinase Src, the phosphatases SHP-1 and SHP-2, and β3 integrin mechanoreceptors. Activation of Src in this complex led to activation of extracellular signal–regulated kinase (ERK), which in turn elicited changes in gene expression that promote proliferation. Thus, PKG-activating drugs could be used to mimic the anabolic effects of mechanical stimulation on bone in the treatment of osteoporosis. The accompanying Perspective by Bidwell and Pavalko describes other examples of signaling pathways that mediate mechanotransduction in bone cells. Mechanical stimulation is crucial for bone growth and remodeling, and fluid shear stress promotes anabolic responses in osteoblasts through multiple second messengers, including nitric oxide (NO). NO triggers production of cyclic guanosine 3′,5′-monophosphate (cGMP), which in turn activates protein kinase G (PKG). We found that the NO-cGMP-PKG signaling pathway activates Src in mechanically stimulated osteoblasts to initiate a proliferative response. PKGII was necessary for Src activation, a process that also required the interaction of Src with β3 integrins and dephosphorylation of Src by a complex containing the phosphatases SHP-1 (Src homology 2 domain–containing tyrosine phosphatase 1) and SHP-2. PKGII directly phosphorylated and stimulated SHP-1 activity, and fluid shear stress triggered the recruitment of PKGII, Src, SHP-1, and SHP-2 to a mechanosome containing β3 integrins. PKGII-null mice showed defective Src and ERK (extracellular signal–regulated kinase) signaling in osteoblasts and decreased ERK-dependent gene expression in bone. Our findings reveal a convergence of NO-cGMP-PKG and integrin signaling and establish a previously unknown mechanism of Src activation. These results support the use of PKG-activating drugs to mimic the anabolic effects of mechanical stimulation of bone in the treatment of osteoporosis.

Impaired platelet function in a patient with P2Y12 deficiency caused by a mutation in the translation initiation codon.

Summary.  In this study, we have identified a patient (OSP‐1) with a congenital P2Y12 deficiency showing a mild bleeding tendency from her childhood and examined the role of P2Y12 in platelet function. At low concentrations of agonists OSP‐1 platelets showed an impaired aggregation to several kinds of stimuli, whereas at high concentrations they showed a specifically impaired platelet aggregation to adenosine diphosphate (ADP). ADP normally induced platelet shape change and failed to inhibit PGE1‐stimulated cAMP accumulation in OSP‐1 platelets. Molecular genetic analysis revealed that OSP‐1 was a homozygous for a mutation in the translation initiation codon (ATG to AGG) in the P2Y12 gene. Heterologous cell expression of wild‐type or mutant P2Y12 confirmed that the mutation was responsible for the deficiency in P2Y12. OSP‐1 platelets showed a markedly impaired platelet spreading onto immobilized fibrinogen. Real‐time observations of thrombogenesis under a high shear rate (2000 s−1) revealed that thrombi over collagen were small and loosely packed and most of the aggregates were unable to resist against high shear stress in OSP‐1. Our data suggest that secretion of endogenous ADP and subsequent P2Y12‐mediated signaling are critical for platelet aggregation, platelet spreading, and as a consequence, for stabilization of thrombus.

The Primacy of β1 Integrin Activation in the Metastatic Cascade

After neoplastic cells leave the primary tumor and circulate, they may extravasate from the vasculature and colonize tissues to form metastases. β1 integrins play diverse roles in tumorigenesis and tumor progression, including extravasation. In blood cells, activation of β1 integrins can be regulated by “inside-out” signals leading to extravasation from the circulation into tissues. However, a role for inside-out β1 activation in tumor cell metastasis is uncertain. Here we show that β1 integrin activation promotes tumor metastasis and that activated β1 integrin may serve as a biomarker of metastatic human melanoma. To determine whether β1 integrin activation can influence tumor cell metastasis, the β1 integrin subunit in melanoma and breast cancer cell lines was stably knocked down with shRNA and replaced with wild-type or constitutively-active β1. When tumor cells expressing constitutively-active β1 integrins were injected intravenously into chick embryos or mice, they demonstrated increased colonization of the liver when compared to cells expressing wild-type β1 integrins. Rescue expression with mutant β1 integrins revealed that tumor cell extravasation and hepatic colonization required extracellular ligand binding to β1 as well as β1 interaction with talin, an intracellular mediator of integrin activation by the Rap1 GTPase. Furthermore, shRNA-mediated knock down of talin reduced hepatic colonization by tumor cells expressing wild-type β1, but not constitutively-active β1. Overexpression in tumor cells of the tumor suppressor, Rap1GAP, inhibited Rap1 and β1 integrin activation as well as hepatic colonization. Using an antibody that detects activated β1 integrin, we found higher levels of activated β1 integrins in human metastatic melanomas compared to primary melanomas, suggesting that activated β1 integrin may serve as a biomarker of invasive tumor cells. Altogether, these studies establish that inside-out activation of β1 integrins promotes tumor cell extravasation and colonization, suggesting diagnostic and therapeutic approaches for targeting of β1 integrin signaling in neoplasia.

Critical role of ADP interaction with P2Y12 receptor in the maintenance of alpha(IIb)beta3 activation: association with Rap1B activation.

Summary.  Objective: Platelet integrin αIIbβ3 plays a crucial role in platelet aggregation, and the affinity of αIIbβ3 for fibrinogen is dynamically regulated. Employing modified ligand‐binding assays, we analyzed the mechanism by which αIIbβ3 maintains its high‐affinity state. Methods and results: Washed platelets adjusted to 50 × 103 μL−1 were stimulated with 0.2 U mL−1 thrombin or 5 μm U46619 under static conditions. After the completion of αIIbβ3 activation and granule secretion, different kinds of antagonists were added to the activated platelets. The activated αIIbβ3 was then detected by fluorescein isothiocyanate (FITC)‐labeled PAC1. The addition of 1 μm AR‐C69931MX (a P2Y12 antagonist) or 1 mm A3P5P (a P2Y1 antagonist) disrupted the sustained αIIbβ3 activation by ∼92% and ∼38%, respectively, without inhibiting CD62P or CD63 expression. Dilution of the platelet preparation to 500 μL−1 also disrupted the sustained αIIbβ3 activation, and the disruption by such dilution was abrogated by the addition of exogenous adenosine 5′‐diphosphate (ADP) in a dose‐dependent fashion. The amounts of ADP released from activated platelets determined by high‐performance liquid chromatography were compatible with the amounts of exogenous ADP required for the restoration. We next examined the effects of antagonists on protein kinase C (PKC) and Rap1B activation induced by 0.2 U mL−1 thrombin. Thrombin induced long‐lasting PKC and Rap1B activation. AR‐C69931MX markedly inhibited Rap1B activation without inhibiting PKC activation. Conclusions: Our data indicate that the continuous interaction between released ADP and P2Y12 is critical for the maintenance of αIIbβ3 activation.

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 others 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.

Interaction Between Src Homology 2 Domain Bearing Protein Tyrosine Phosphatase Substrate-1 and CD47 Mediates the Adhesion of Human B Lymphocytes to Nonactivated Endothelial Cells

CD47 modulates a variety of cell functions such as adhesion, spreading, and migration. Using a fusion protein consisting of the extracellular region of Src homology 2 domain bearing protein tyrosine phosphatase substrate-1 (SHPS-1) and the Fc portion of human Ig (SHPS-1-Ig) we investigated the effects of SHPS-1 as a ligand for CD47 on B lymphocytes. Although SHPS-1-Ig binding to human B cell lines was solely mediated via CD47, their binding capacity for soluble and immobilized SHPS-1-Ig varied among cell lines irrespective of the similar expression levels of CD47, suggesting that distinctive affinity/avidity states exist during B cell maturation. Nalm6 cell line and tonsilar B lymphocytes adhered to immobilized SHPS-1-Ig and showed polarization-like morphology. These effects of SHPS-1-Ig were blocked by anti-CD47 mAbs (B6H12 and SE5A5). Wortmannin, a phosphatidylinositol-3 kinase inhibitor, but not pertussis toxin significantly inhibited the polarization induced by the immobilized SHPS-1-Ig. Thus, SHPS-1 acts as an adhesive substrate via CD47 in human B lymphocyte. Immunohistochemical analyses indicated that SHPS-1 is expressed on high endothelial venule as well as macrophages in human tonsils. HUVECs also express SHPS-1 in the absence of any stimuli, and the adhesion of tonsilar B lymphocytes to nonactivated HUVECs was significantly inhibited by SE5A5, indicating that SHPS-1/CD47 interaction is involved in the adhesion. Our findings suggest that SHPS-1/CD47 interaction may contribute to the recruitment of B lymphocytes via endothelial cells under steady state conditions.

Differences in Regulation of Drosophila and Vertebrate Integrin Affinity by Talin

Integrin-mediated cell adhesion is essential for development of multicellular organisms. In worms, flies, and vertebrates, talin forms a physical link between integrin cytoplasmic domains and the actin cytoskeleton. Loss of either integrins or talin leads to similar phenotypes. In vertebrates, talin is also a key regulator of integrin affinity. We used a ligand-mimetic Fab fragment, TWOW-1, to assess talins role in regulating Drosophila alphaPS2 betaPS affinity. Depletion of cellular metabolic energy reduced TWOW-1 binding, suggesting alphaPS2 betaPS affinity is an active process as it is for vertebrate integrins. In contrast to vertebrate integrins, neither talin knockdown by RNA interference nor talin head overexpression had a significant effect on TWOW-1 binding. Furthermore, replacement of the transmembrane or talin-binding cytoplasmic domains of alphaPS2 betaPS with those of human alphaIIb beta3 failed to enable talin regulation of TWOW-1 binding. However, substitution of the extracellular and transmembrane domains of alphaPS2 betaPS with those of alphaIIb beta3 resulted in a constitutively active integrin whose affinity was reduced by talin knockdown. Furthermore, wild-type alphaIIb beta3 was activated by overexpression of Drosophila talin head domain. Thus, despite evolutionary conservation of talins integrin/cytoskeleton linkage function, talin is not sufficient to regulate Drosophila alphaPS2 betaPS affinity because of structural features inherent in the alphaPS2 betaPS extracellular and/or transmembrane domains.

Identification of amino-terminal region of adiponectin as a physiologically functional domain

Adiponectin is an abundant adipose‐specific protein, which acts as an anti‐diabetic, anti‐atherogenic, and anti‐inflammatory adipokine. Although recent advances in the field of adiponectin have been made by the identification of adiponectin receptors and by the understanding about relationship between its multimerization and functions, detailed molecular background remains unclear. Our established anti‐human adiponectin antibodies, ANOC 9103 and ANOC 9104, blocked some adiponectin functions such as the growth inhibition of B‐lymphocytes on stromal cells and the inhibition of acetylated LDL uptake in macrophages, suggesting that they may recognize important functional regions of adiponectin. As a result of epitope mapping based on the ability to bind to the deleted adiponectin mutants, we identified that these antibodies recognize amino‐terminal region of adiponectin before the beginning of the collagen‐like domain. Notably, a peptide fragment (DQETTTQGPGVLLPLPKGACTGWMA) corresponding to amino acid residues 17–41 of human adiponectin could bind to restricted types of cells and block adiponectin‐induced cyclooxygenase‐2 gene expression and prostaglandin E2 production in MS‐5 stromal cells. Moreover, the deletion of its amino‐terminal region reduced the abilities to inhibit not only collagen‐induced platelet aggregation but also diet‐induced hepatic steatosis. These data indicate that amino‐terminal region of adiponectin is a physiologically functional domain and that a novel receptor, which recognizes amino‐terminal region of adiponectin, may exist on some types of cells. Further investigations will contribute to the understanding of molecular mechanisms about adiponectin functions as well as to the designing of novel strategies for the treatment of patients with insulin‐resistance, vascular dysfunction, and chronic inflammation. J. Cell. Biochem. 98: 194–207, 2006.