Biomedical subjects

Stephen C-T Lam

Publications and source records attributed to Stephen C-T Lam.

11 recordsLinked to original sources

PubMed2006

TA205, an anti-talin monoclonal antibody, inhibits integrin-talin interaction.

Talin mediates integrin signaling by binding to integrin cytoplasmic tails through its FERM domain which consists of F1, F2 and F3 subdomains. TA205, an anti-talin monoclonal antibody, disrupts actin stress fibers and focal adhesion when microinjected into fibroblasts. Here, we showed that TA205 caused an allosteric inhibition of integrin alphaIIb beta3 binding to the talin FERM domain and mapped the TA205 epitope to residues 131-150 in talin F1. Furthermore, binding of a talin rod fragment to talin head was partially inhibited by TA205. These findings suggest that talin F1 may be important in regulation of integrin binding and talin head-rod interaction.

Allosteric Regulation↗

PubMed2006

CIB1 is an endogenous inhibitor of agonist-induced integrin alphaIIbbeta3 activation.

In response to agonist stimulation, the alphaIIbbeta3 integrin on platelets is converted to an active conformation that binds fibrinogen and mediates platelet aggregation. This process contributes to both normal hemostasis and thrombosis. Activation of alphaIIbbeta3 is believed to occur in part via engagement of the beta3 cytoplasmic tail with talin; however, the role of the alphaIIb tail and its potential binding partners in regulating alphaIIbbeta3 activation is less clear. We report that calcium and integrin binding protein 1 (CIB1), which interacts directly with the alphaIIb tail, is an endogenous inhibitor of alphaIIbbeta3 activation; overexpression of CIB1 in megakaryocytes blocks agonist-induced alphaIIbbeta3 activation, whereas reduction of endogenous CIB1 via RNA interference enhances activation. CIB1 appears to inhibit integrin activation by competing with talin for binding to alphaIIbbeta3, thus providing a model for tightly controlled regulation of alphaIIbbeta3 activation.

Animals↗

PubMed2004

Targeted mutagenesis of the angiogenic protein CCN1 (CYR61). Selective inactivation of integrin alpha6beta1-heparan sulfate proteoglycan coreceptor-mediated cellular functions.

The matricellular protein CCN1 (CYR61) regulates multiple cellular processes and plays essential roles in embryonic vascular development. A ligand of several integrin receptors, CCN1 acts through integrin alpha6beta1 and heparan sulfate proteoglycans (HSPGs) to promote specific functions in fibroblasts, smooth muscle cells, and endothelial cells. We have previously identified a novel alpha6beta1 binding site, T1, in domain III of CCN1. Here we uncover two novel 16-residue sequences, H1 and H2, in domain IV that can support alpha6beta1- and HSPGs-dependent cell adhesion, suggesting that these sequences contain closely juxtaposed or overlapping sites for interaction with alpha6beta1 and HSPGs. Furthermore, fibroblast adhesion to the H1 and H2 peptides is sufficient to induce prolonged MAPK activation, whereas adhesion to T1 induces transient MAPK activation. To dissect the roles of these sites in CCN1 function, we have created mutants disrupted in T1, H1, and H2 or in all three sites in the context of full-length CCN1. We show that the T1 and H1/H2 sites are functionally non-equivalent, and disruption of these sites differentially affected cell adhesion, migration, mitogen-activated protein kinase activation, and regulation of gene expression. Disruption of all three sites completely abolished alpha6beta1-HSPG-mediated cellular activities. All mutants disrupting T1, H1, and H2 fully retain alphavbeta3-mediated pro-angiogenic activities, indicating that these mutants are biologically active and are defective only in alpha6beta1-HSPG-mediated functions. Together, these findings identify and dissect the differential roles of the three sites (T1, H1, H2) required for alpha6beta1-HSPG-dependent CCN1 activities and provide a strategy to investigate these alpha6beta1-HSPG-specific activities in vivo.

Amino Acid Sequence↗

PubMed2004

Identification of a novel integrin alphavbeta3 binding site in CCN1 (CYR61) critical for pro-angiogenic activities in vascular endothelial cells.

CCN1 (CYR61) is a matricellular inducer of angiogenesis essential for successful vascular development. Though devoid of the canonical RGD sequence motif recognized by some integrins, CCN1 binds to, and functions through integrin alphavbeta3 to promote pro-angiogenic activities in activated endothelial cells. In this study we identify a 20-residue sequence, V2 (NCKHQCTCIDGAVGCIPLCP), in domain II of CCN1 as a novel binding site for integrin alphavbeta3. Immobilized synthetic V2 peptide supports alphavbeta3-mediated cell adhesion; soluble V2 peptide inhibits endothelial cell adhesion to CCN1 and the homologous family members CCN2 (connective tissue growth factor, CTGF) or CCN3 (NOV) but not to collagen. These activities are obliterated by mutation of the aspartate residue in the V2 peptide to alanine. The corresponding D125A mutation in the context of the N-terminal half of CCN1 (domains I and II) greatly diminished direct solid phase binding to purified integrin alphavbeta3 and abolished alphavbeta3-mediated cell adhesion activity. Likewise, soluble full-length CCN1 with the D125A mutation is defective in binding purified alphavbeta3 and impaired in alphavbeta3-mediated pro-angiogenic activities in vascular endothelial cells, including stimulation of cell migration and enhancement of DNA synthesis. In contrast, immobilized full-length CCN1-D125A mutant binds alphavbeta3 and supports alphavbeta3-mediated cell adhesion similar to wild type CCN1. These results indicate that V2 is the primary alphavbeta3 binding site in soluble CCN1, whereas additional cryptic alphavbeta3 binding site(s) in the C-terminal half of CCN1 becomes exposed when the protein is immobilized. Together, these results identify a novel and functionally important binding site for integrin alphavbeta3 and provide a new approach for dissecting alphavbeta3-specific CCN1 functions both in cultured cells and in the organism.

Amino Acid Sequence↗

PubMed2003

Critical roles for the COOH-terminal NITY and RGT sequences of the integrin beta3 cytoplasmic domain in inside-out and outside-in signaling.

Bidirectional signaling of integrin alphaIIbbeta3 requires the beta3 cytoplasmic domain. To determine the sequence in the beta3 cytoplasmic domain that is critical to integrin signaling, cell lines were established that coexpress the platelet receptor for von Willebrand factor (vWF), glycoprotein Ib-IX, integrin alphaIIb, and mutants of beta3 with truncations at sites COOH terminal to T741, Y747, F754, and Y759. Truncation at Y759 did not affect integrin activation, as indicated by vWF-induced fibrinogen binding, but affected cell spreading and stable adhesion. Thus, the COOH-terminal RGT sequence of beta3 is important for outside-in signaling but not inside-out signaling. In contrast, truncation at F754, Y747, or T741 completely abolished integrin activation. A point mutation replacing Y759 with alanine also abolished integrin activation. Thus, the T755NITY759 sequence of beta3, containing an NXXY motif, is critical to inside-out signaling, whereas the intact COOH terminus is important for outside-in signaling. In addition, we found that the calcium-dependent protease calpain preferentially cleaves at Y759 in a population of beta3 during platelet aggregation and adhesion, suggesting that calpain may selectively regulate integrin outside-in signaling.

Alanine↗

PubMed2003

Identification of a novel integrin alpha 6 beta 1 binding site in the angiogenic inducer CCN1 (CYR61).

The angiogenic inducer CCN1 (cysteine-rich 61, CYR61), a secreted matricellular protein of the CCN family, is a ligand of multiple integrins, including alpha 6 beta 1. Previous studies have shown that CCN1 interaction with integrin alpha 6 beta 1 mediates adhesion of fibroblasts, endothelial cells, and smooth muscle cells, as well as migration of smooth muscle cells. Recently, we have reported that CCN1-induced tubule formation of unactivated endothelial cells is also mediated through integrin alpha 6 beta 1. In this study, we demonstrate that human skin fibroblasts adhere specifically to the T1 sequence (GQKCIVQTTSWSQCSKS) within domain III of CCN1, and this process is blocked by anti-alpha 6 and anti-beta 1 monoclonal antibodies. Alanine substitution mutagenesis of the T1 sequence further defines the sequence TTSWSQCSKS as the critical determinant for mediating alpha 6 beta 1-dependent adhesion. Soluble T1 peptide specifically inhibits fibroblast adhesion to CCN1 in a dose-dependent manner. Furthermore, T1 also inhibits cell adhesion to other alpha 6 beta 1 ligands, including CCN2 (CTGF), CCN3 (NOV), and laminin, but not to ligands of other integrins. In addition, T1 specifically inhibits alpha 6 beta 1-dependent tubule formation of unactivated endothelial cells in a CCN1-containing collagen gel matrix. To confirm that T1 binds integrin alpha 6 beta 1 directly, we perform affinity chromatography and show that integrin alpha 6 beta 1 is isolated from an octylglucoside extract of fibroblasts on T1-coupled Affi-gel. Taken together, these findings define the T1 sequence in CCN1 as a novel binding motif for integrin alpha 6 beta 1, providing the basis for the development of peptide mimetics to examine the functional role of alpha 6 beta 1 in angiogenesis.

Alanine↗

PubMed2003

Identification of a novel integrin alphaMbeta2 binding site in CCN1 (CYR61), a matricellular protein expressed in healing wounds and atherosclerotic lesions.

CCN1 (cysteine-rich 61) and CCN2 (connective tissue growth factor) are growth factor-inducible immediate-early gene products found in atherosclerotic lesions, restenosed blood vessels, and healing cutaneous wounds. Both CCN proteins have been shown to support cell adhesion and induce cell migration through interaction with integrin receptors. Recently, we have identified integrin alphaMbeta2 as the major adhesion receptor mediating monocyte adhesion to CCN1 and CCN2 and have shown that the alphaMI domain binds specifically to both proteins. In the present study, we demonstrated that activated monocytes adhered to a synthetic peptide (CCN1-H2, SSVKKYRPKYCGS) derived from a conserved region within the CCN1 C-terminal domain, and this process was blocked by the anti-alphaM monoclonal antibody 2LPM19c. Consistently, a glutathione S-transferase (GST) fusion protein containing the alphaMI domain (GST-alphaMI) bound to immobilized CCN1-H2 as well as to the corresponding H2 sequence in CCN2 (CCN2-H2, TSVKTYRAKFCGV). By contrast, a scrambled CCN1-H2 peptide and an 18-residue peptide derived from an adjacent sequence of CCN1-H2 failed to support monocyte adhesion or alphaMI domain binding. To confirm that the CCN1-H2 sequence within the CCN1 protein mediates alphaMbeta2 interaction, we developed an anti-peptide antibody against CCN1-H2 and showed that it specifically blocked GST-alphaMI binding to intact CCN1. Collectively, these results identify the H2 sequence in CCN1 and CCN2 as a novel integrin alphaMbeta2 binding motif that bears no apparent homology to any alphaMbeta2 binding sequence reported to date.

Amino Acid Motifs↗

PubMed2002

A molecular basis for integrin alphaMbeta 2 ligand binding promiscuity.

The leukocyte integrin alpha(M)beta(2) is a highly promiscuous leukocyte receptor capable of binding a multitude of unrelated ligands. To understand the molecular basis for the broad ligand recognition of alpha(M)beta(2), the inter-integrin chimera was created. In the chimeric integrin, the betad-alpha5 loop-alpha5 helix segment comprised of residues Lys(245)-Arg(261) from the alpha(M)I domain of alpha(M)beta(2) was inserted into the framework of alpha(L)beta(2). The construct was expressed in HEK 293 cells, and the ability of generated cells to adhere to fibrinogen and its derivatives was characterized first. Grafting the alpha(M)(Lys(245)-Arg(261)) sequence converted alpha(L)beta(2) into a fibrinogen-binding protein capable of mediating efficient and specific adhesion similar to that of wild-type alpha(M)beta(2). Verifying a switch in the binding specificity of alpha(L)beta(2), the chimeric receptor became competent to support cell migration to fibrinogen. Mutations at positions Phe(246), Asp(254), and Pro(257) within Lys(245)-Arg(261) of alpha(M)beta(2) produced significant decreases in cell adhesion, illustrating the critical role of these residues in ligand binding. The insertion of alpha(M)(Lys(245)-Arg(261)) imparted to the chimeric integrin the ability to recognize many typical alpha(M)beta(2) protein ligands. Furthermore, cells expressing the chimeric receptor, but not alpha(L)beta(2), were able to stick to uncoated plastic, which represents the hallmark of wild-type alpha(M)beta(2). These results suggest that alpha(M)(Lys(245)-Arg(261)) serves as a consensus binding site for interaction with a variety of distinct molecules and, thus, may define the degenerate recognition properties inherent to alpha(M)beta(2).

Amino Acid Sequence↗

PubMed2002

Pro-angiogenic activities of CYR61 (CCN1) mediated through integrins alphavbeta3 and alpha6beta1 in human umbilical vein endothelial cells.

CYR61 (CCN1) is an extracellular matrix-associated protein of the CCN family, which also includes CTGF (CCN2), NOV (CCN3), WISP-1 (CCN4), WISP-2 (CCN5), and WISP-3 (CCN6). Purified CYR61 induces neovascularization in corneal implants, and Cyr61-null mice suffer embryonic death due to vascular defects, thus establishing that CYR61 is an important regulator of angiogenesis. Aberrant expression of Cyr61 is associated with breast cancer, wound healing, and vascular diseases such as atherosclerosis and restenosis. In culture, CYR61 functions through integrin-mediated pathways to promote cell adhesion, migration, and proliferation. Here we show that CYR61 can also promote cell survival and tubule formation in human umbilical vein endothelial cells. Furthermore, we have dissected the integrin receptor requirements of CYR61 with respect to its pro-angiogenic activities. Thus, CYR61-induced cell adhesion and tubule formation occur through interaction with integrin alpha(6)beta(1) in early passage endothelial cells in which integrins have not been activated. By contrast, in endothelial cells in which integrins are activated by phorbol ester or vascular endothelial growth factor, CYR61-promoted cell adhesion, migration, survival, growth factor-induced mitogenesis, and endothelial tubule formation are all mediated through integrin alpha(v)beta(3). These findings indicate that CYR61 is an activation-dependent ligand of integrin alpha(v)beta(3) and an activation-independent ligand of integrin alpha(6)beta(1) and that these integrins differentially mediate the pro-angiogenic activities of CYR61. These findings help to define the mechanisms by which CYR61 acts as an angiogenic regulator, provide a molecular interpretation for the loss of vascular integrity and increased apoptosis of vascular cells in Cyr61-null mice, and underscore the importance of CYR61 in the development and homeostasis of the vascular system.

Cell Adhesion↗

PubMed2002

Identification of integrin alpha(M)beta(2) as an adhesion receptor on peripheral blood monocytes for Cyr61 (CCN1) and connective tissue growth factor (CCN2): immediate-early gene products expressed in atherosclerotic lesions.

Cysteine-rich 61 (Cyr61, CCN1) and connective tissue growth factor (CTGF, CCN2) are growth factor-inducible immediate-early gene products found in blood vessel walls and healing cutaneous wounds. We previously reported that the adhesion of endothelial cells, platelets, and fibroblasts to these extracellular matrix-associated proteins is mediated through integrin receptors. In this study, we demonstrated that both Cyr61 and CTGF are expressed in advanced atherosclerotic lesions of apolipoprotein E-deficient mice. Because monocyte adhesion and transmigration are important for atherosclerosis, wound healing, and inflammation, we examined the interaction of THP-1 monocytic cells and isolated peripheral blood monocytes with Cyr61 and CTGF. THP-1 cells and monocytes adhered to Cyr61- or CTGF-coated wells in an activation-dependent manner and this process was mediated primarily through integrin alpha(M)beta(2). Additionally, expression of alpha(M)beta(2) on human embryonic kidney 293 cells resulted in enhanced cell adhesion to Cyr61. Consistent with these data, a GST-fusion protein containing the I domain of the integrin alpha(M) subunit bound specifically to immobilized Cyr61 or CTGF. We have also investigated the requirement of cell surface heparan sulfate proteoglycans (HSPGs) as coreceptors for monocyte adhesion to Cyr61. Pretreatment of monocytes with heparin or heparinase I resulted in partial inhibition of cell adhesion to Cyr61. However, monocytes, but not fibroblasts, were capable of adhering to a Cyr61 mutant deficient in heparin binding activity. Collectively, these results show that activated monocytes adhere to Cyr61 and CTGF through integrin alpha(M)beta(2) and cell surface HSPGs. However, unlike fibroblast adhesion to Cyr61, cell surface HSPGs are not absolutely required for this adhesion process.

Animals↗

PubMed2002

The angiogenic factor cysteine-rich 61 (CYR61, CCN1) supports vascular smooth muscle cell adhesion and stimulates chemotaxis through integrin alpha(6)beta(1) and cell surface heparan sulfate proteoglycans.

Cysteine-rich 61 (CYR61, CCN1) is a heparin-binding, extracellular, matrix-associated protein of the cysteine-rich 61/nephroblastoma family, which also includes connective tissue growth factor, nephroblastoma overexpressed, Wnt-induced secreted protein-1 (WISP-1), WISP-2, and WISP-3. CYR61 induces angiogenesis in vivo and supports cell adhesion, promotes cell migration, and enhances growth factor-stimulated mitogenesis in fibroblasts and endothelial cells. Although the expression of CYR61 has been observed in arterial walls, its function in vascular smooth muscle cells (VSMCs) has not been examined to date. Here we show that purified CYR61 supports VSMC adhesion in a dose-dependent, saturable manner through integrin alpha(6)beta(1) with an absolute requirement of cell surface heparan sulfate proteoglycans. In addition, CYR61 induces VSMC chemotaxis, but not chemokinesis, through integrin alpha(6)beta(1) and heparan sulfate proteoglycans. Heparin-binding defective CYR61 mutants are unable to support VSMC adhesion but can still induce chemotaxis at a reduced level. Following balloon angioplasty in rat carotid artery, CYR61 protein level is elevated in the media and neointima of the injured vessel by d 4 post angioplasty, peaks from d 7 to 14, and remains high for at least 28 d. These data demonstrate the activities of CYR61 in VSMCs, identify the receptors that mediate its functions, and show that CYR61 is synthesized in arterial smooth muscle walls during proliferative restenosis. Together, these results implicate CYR61 as a novel factor that modulates the responses of VSMCs to vascular injury.

Angioplasty, Balloon↗