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Regulation of Tumor Cell Chemotaxis by Type IV Collagen Is Mediated by a Ca2+-dependent Mechanism Requiring CD47 and the Integrin αVβ3*

Open AccessPublished:February 18, 2000DOI:https://doi.org/10.1074/jbc.275.7.4796
      Studies from our laboratories demonstrated that synthetic peptides from the non-collagenous (NC-1) domain of the α3 (IV) chain of type IV collagen (COL IV) enhanced tumor cell adhesion (Han, J., Ohno, N., Monboisse, J. C., Pasco, S., Borel, J. P., and Kefalides, N. A. (1997) J. Biol. Chem. 272, 20395–20401). We have isolated the receptors for the α3(IV)185–203 peptide from melanoma and prostate tumor cells and identified them as CD47/integrin-associated protein and the integrin αVβ3 (Shahan, T. A., Ziaie, Z., Pasco, S., Fawzi, A., Bellon, G., Monboisse, J. C., and Kefalides, N. A. (1999) Cancer Res. 59, 4584–4590). In the present study we have examined the effect of CD47 and the integrin αVβ3 on in vitro tumor cell chemotaxis and Ca2+imodulation in response to COL IV, from the anterior lens capsule (ALC-COL IV) and peptides from its NC-1 domain. COL IV as well as the α3(IV) peptide promoted tumor cell chemotaxis with an immediate increase in intracellular [Ca2+]. Treating tumor cells with CD47 and integrin αVβ3-reactive antibodies reduced chemotaxis as well as the rise in [Ca2+]i in response to ALC-COL IV or the α3(IV)185–203 peptide but not to Engelbreth-Holm-Swarm-COL IV or fibronectin. The α3(IV)185–203 synthetic peptide stimulated an increase in calcium from intracellular stores exclusively, whereas ALC-COL IV, Engelbreth-Holm-Swarm-COL IV, and fibronectin stimulated Ca2+ flux from both internal and external stores. Furthermore, treatment of the cells with Ca2+ chelator bis-(O-aminophenoxyl)ethane-N,N,N′,N′-tetraaceticacid-acetomethoxy ester inhibited chemotaxis toward both ALC-COL IV and the α3(IV)185–203 peptide. These data indicate that CD47 and integrin αVβ3 regulate tumor cell chemotaxis in response to COL IV and the α3(IV)185–203 peptide through a Ca2+-dependent mechanism.
      BM
      basement membrane
      ALC-COL IV
      type IV collagen from anterior lens capsule
      BAPTA-AM
      bis-(O-aminophenoxyl)ethane-N,N,N′,N′-tetraacetic acid-acetomethoxy ester
      IAP
      integrin-associated protein
      COL IV
      type IV collagen
      EHS
      Engelbreth-Holm-Swarm tumor
      FBS
      fetal bovine serum
      HBSS
      Hanks' balanced salt solution
      i
      intracellular
      mAb
      monoclonal antibody
      NC-1
      noncollagenous domain
      HPF
      high power field
      PMN
      polymorphonuclear leukocyte
      Transmigration through the vessel wall by tumor cells requires adhesion and penetration of the basement membrane (BM),1 followed by movement in or out of the vessel lumen. These processes are mediated through specific adhesion molecules on tumor cells and endothelial cells (
      • Oura E.B.
      • Sandig M.
      • Siu C.H.
      ,
      • Akiyama S.K.
      • Olden K.
      • Yamada K.M.
      ). Integrins play an important role in cellular adhesion and motility by allowing cellular adhesion and activation of signal transduction pathways (,
      • Albelda S.M.
      • Buck C.A.
      ).
      Normal COL IV is a triple-helical molecule formed by the interaction of any two of six different α-chains (α1–α6) (
      • Gunwar S.
      • Saus J.
      • Noelken M.E.
      • Hudson B.G.
      ,
      • Hudson B.G.
      • Reeders S.T.
      • Tryggvason K.
      ,
      • Leinonen A.
      • Mariyana M.
      • Mochizuki T.
      • Tryggvason K.
      • Reeders S.T.
      ). We have shown that synthetic peptides comprising residues 185–203 of the NC-1 domain of the α3 chain of COL IV from anterior lens capsule (ALC), enhance melanoma cell adhesion by 50–60% over controls. On the other hand, peptides from a similar region of the other chains (i.e.α1, α2, α4, or α5) were less efficient in promoting adhesion. In these biological activities, the presence of the -SNS- triplet (residues 189–191) in the above peptide appears to be an absolute requirement (
      • Han J.
      • Ohno N.
      • Monboisse J.C.
      • Pasco S.
      • Borel J.P.
      • Kefalides N.A.
      ). Pretreatment of the α3(IV)185–203 peptide substrate with a peptide-reactive monoclonal antibody (mAb) inhibited melanoma cell attachment; however, the treatment of a substrate composed of EHS-COL IV did not inhibit attachment.
      More recently we have isolated the α3(IV)185–203 peptide-specific receptors on melanoma and prostate tumor cells and identified them as CD47/integrin-associated protein and the αVβ3 integrin by affinity chromatography and Western blot analysis (
      • Shahan T.A.
      • Ziaie Z.
      • Pasco S.
      • Fawzi A.
      • Bellon G.
      • Monboisse J.C.
      • Kefalides N.A.
      ). COL IV from the Engelbreth-Holm-Swarm (EHS) mouse tumor, which contains only the α1 and α2 chains of COL IV (
      • Wisdom Jr., B.J.
      • Gunwar S.
      • Hudson M.D.
      • Noelken M.E.
      • Hudson B.G.
      ) has been previously demonstrated to induce melanoma cell chemotaxis (
      • Savarese D.M.
      • Russell J.T.
      • Fatatis A.
      • Liotta L.
      ). The role of CD47 in tumor cell chemotaxis has never been explored; however, Cooper et al. (
      • Cooper D.
      • Lindberg F.P.
      • Gamble J.R.
      • Brown E.J.
      • Vadas M.A.
      ) and Parkoset al. (
      • Parkos C.A.
      • Colgan S.P.
      • Liang T.W.
      • Nusrat A.
      • Bacarra A.E.
      • Carnes D.K.
      • Madara J.L.
      ) demonstrated the requirement of CD47 for normal trans-endothelial and trans-epithelial migration of PMN. Work by Parkoset al. (
      • Parkos C.A.
      • Colgan S.P.
      • Liang T.W.
      • Nusrat A.
      • Bacarra A.E.
      • Carnes D.K.
      • Madara J.L.
      ) showed that PMN, pretreated with CD47-reactive antibodies at levels above 2 μg/ml, were inhibited from trans-endothelial migration in response ton-formylmethionylleucylphenylalanine. They concluded that the inhibition was due to a yet unknown post-adhesive event following binding to the β2 integrin subunit.
      The signal transduction pathways by which CD47 and integrin αVβ3 regulate chemotaxis are mostly unknown. Schwartz et al. (
      • Schwartz M.A.
      • Brown E.J.
      • Fazeli B.
      ) first described the role of CD47 on Ca2+ flux in endothelial cells. Based on the primary sequence homology of CD47 with known Ca2+ channel proteins, they hypothesized that it may play a role in Ca2+modulation. Their data showed that CD47 is specifically required for integrin-mediated Ca2+ flux in endothelial cells (
      • Schwartz M.A.
      • Brown E.J.
      • Fazeli B.
      ). The role of Ca2+ modulation in the alteration of the cell cytoskeleton to accommodate cell movement for spreading and motility has been demonstrated (
      • Smith T.W.
      • Menter D.G.
      • Nicholson G.L.
      • McIntire L.V.
      ,
      • Cunningham C.C.
      • Gorlin J.B.
      • Kwiatkowski D.J.
      • Hartwig J.H.
      • Janmey P.A.
      • Byers H.R.
      • Stossel T.P.
      ). However, the effect of CD47 on the regulation of Ca2+ flux and motility in tumor cells has not been previously reported.
      The primary aim of this study was to examine the influence of CD47 and integrin αVβ3 on tumor cell chemotaxis in response to COL IV and the NC-1 domain of its α3 chain. The data do indicate that both CD47 and integrin αVβ3influence tumor cell chemotaxis in response to COL IV and to synthetic peptides from the α3(IV) chain. Furthermore, we show that the α3(IV)185–203 peptide causes an immediate rise in [Ca2+]i that was inhibited by pretreatment of tumor cells with CD47 or integrin αVβ3-reactive mAbs. The Ca2+chelator BAPTA-AM inhibited chemotaxis toward COL IV, indicating an association between the receptors, cell motility, and Ca2+flux in response to COL IV and its peptides.

      DISCUSSION

      The ability of CD47 and integrin αVβ3to function as receptors mediating chemotaxis in response to the α3 chain of COL IV is a novel observation. Type IV collagen is found in BM and contributes to its structural and functional properties. Our studies have discovered additional biological properties of BMs that can be attributed to COL IV. Because of their critical location, underlying all endothelial and epithelial surfaces, BMs can be considered as our first line of defense. As with the enhancement of PMN chemotaxis toward BM components (
      • Eble J.A.
      • Ries A.
      • Lichy A.
      • Mann K.
      • Stanton H.
      • Gavrilovic J.
      • Murphy G.
      • Kuhn K.
      ), the present studies demonstrate that COL IV and the α3(IV)185–203 peptide enhance tumor cell chemotaxis. The chemotactic potential of other BM components such as fibronectin, laminin, and entactin has also been demonstrated (
      • Senior R.M.
      • Gresham H.D.
      • Griffin G.L.
      • Brown E.J.
      • Chung A.E.
      ,
      • Klominek J.
      • Sumitran-Karuppan S.
      • Hauzenberger D.
      ).
      In previous studies, we demonstrated that the α3(IV)185–203 peptide bound specifically to CD47 and the αVβ3integrin (
      • Shahan T.A.
      • Ziaie Z.
      • Pasco S.
      • Fawzi A.
      • Bellon G.
      • Monboisse J.C.
      • Kefalides N.A.
      ). CD47 is known to influence PMN motility; however, the mechanism of this process is unknown (
      • Cooper D.
      • Lindberg F.P.
      • Gamble J.R.
      • Brown E.J.
      • Vadas M.A.
      ,
      • Parkos C.A.
      • Colgan S.P.
      • Liang T.W.
      • Nusrat A.
      • Bacarra A.E.
      • Carnes D.K.
      • Madara J.L.
      ). In this report, we present evidence which demonstrates that these receptors are necessary for unabated tumor cell chemotaxis toward COL IV and the α3(IV)185–203 peptide. Treatment of these cells with either CD47 or integrin αVβ3 mAbs alone partially inhibited chemotaxis; however, treatment of the tumor cells with both mAbs together markedly inhibited chemotaxis toward the α3(IV)185–203 peptide and ALC-COL IV. On the other hand, the mAbs had little or no effect in the presence of other chemoattractants such as EHS-COL IV or fibronectin. These data indicate that CD47 and integrin αVβ3 are involved in chemotaxis in response to ALC-COL IV and the α3(IV)185–203 peptide.
      CD47 has been previously shown to associate with and modulate integrin activation. Data support its association with the β1(
      • Vandenberg P.
      • Kern A.
      • Ries A.
      • Luckenbill-Edds L.
      • Mann K.
      • Kuhn K.
      ), β2 (
      • Senior R.M.
      • Gresham H.D.
      • Griffin G.L.
      • Brown E.J.
      • Chung A.E.
      ,
      • Klominek J.
      • Sumitran-Karuppan S.
      • Hauzenberger D.
      ), and β3 (
      • Wang X.Q.
      • Frazier W.A.
      ,
      • Van Strijp J.A.
      • Russell D.G.
      • Tuomanen E.
      • Brown E.J.
      • Wright S.D.
      ,
      • Ishibashi Y.
      • Claus S.
      • Relman D.A
      ) integrin subunits. Previous data from our laboratory suggest that the interaction of CD47 with αVβ3 is necessary for the complete inhibition of tumor cell proliferation and normal tumor cell adhesion to a substrate composed of the α3(IV)185–203 peptide (
      • Shahan T.A.
      • Ziaie Z.
      • Pasco S.
      • Fawzi A.
      • Bellon G.
      • Monboisse J.C.
      • Kefalides N.A.
      ). In the present study, we have presented evidence that both these receptors are also necessary for normal chemotaxis toward the α3(IV)185–203 peptide. The same argument holds true for the rise in [Ca2+]i in response to the above peptide.
      The requirement of Ca2+ flux for cell movement has already been described (
      • Smith T.W.
      • Menter D.G.
      • Nicholson G.L.
      • McIntire L.V.
      ,
      • Cunningham C.C.
      • Gorlin J.B.
      • Kwiatkowski D.J.
      • Hartwig J.H.
      • Janmey P.A.
      • Byers H.R.
      • Stossel T.P.
      ). Our data demonstrate that COL IV and the α3(IV)185–203 peptide cause an immediate, unsustained rise in [Ca2+]i. Furthermore, treatment of melanoma cells with either CD47 or integrin αVβ3-reactive mAbs alone inhibited the rise in [Ca2+]i (Fig. 5, Aand B). In comparison, treatment of melanoma cells with either CD47 or integrin αVβ3-reactive mAbs alone inhibited chemotaxis partially in response to ALC-COL IV and markedly in response to the α3(IV)185–203 peptide but had no effect on chemotaxis toward EHS-COL IV. These data suggest that the rise in [Ca2+]i in response to the α3(IV)185–203 peptide is mediated through the same receptors for the peptide as those required for chemotaxis.
      Based on the predicted structure of CD47 (5 transmembrane domains) and its primary sequence homology to known channel proteins, it has been hypothesized that the receptor may function in the capacity of an ion channel or be closely associated with one (
      • Savarese D.M.
      • Russell J.T.
      • Fatatis A.
      • Liotta L.
      ). To further investigate the relationship among these factors, namely the peptide, the two receptors, and Ca2+ modulation, we examined the source of the Ca2+ flux. We found that the rise in [Ca2+]i in response to the α3(IV)185–203 peptide was primarily from intracellular sources. In comparison, the rise in [Ca2+]i in response to ALC-COL IV and EHS-COL IV was mostly from extracellular sources. Since we have demonstrated that the receptors for the α3(IV)185–203 peptide are CD47 and integrin αVβ3, these data suggest that CD47 may not act as an ion channel in melanoma cells in response to the α3(IV)185–203 peptide; however, the receptors are likely linked to a signal transduction pathway that leads to the release of intracellular Ca2+ stores. On the other hand, treatment of melanoma cells with the CD47 mAb (B6H12) at concentrations above 10 μg/ml inhibited the rise in [Ca2+]i in response to either EHS-COL IV and fibronectin, each of which were shown to cause a rise in [Ca2+]i mostly from extracellular stores. This indicates that CD47 may function as a Ca2+ channel in response to certain agents, but not in response to the α3(IV)185–203 peptide.
      In this report we have demonstrated a regulatory effect of normal type IV collagen from ALC and peptides derived from the NC-1 domain of the α3(IV) chain on tumor cell chemotaxis in vitro. Specifically, we have shown that CD47 and the αVβ3 integrin are necessary for melanoma cell chemotaxis in response to the α3(IV)185–203 peptide. Our current findings also indicate that calcium flux is an important regulator of chemotaxis toward these ligands and suggest the presence of a Ca2+-sensitive signal transduction pathway through CD47 and the integrin αVβ3.

      Acknowledgments

      We thank Dr. Hynda Kleinman for the EHS-type IV collagen and Dr. Eric Brown for initially providing us with the OV10 cell lines and the CD47-reactive mAb (B6H12). We also thank Dr. Caroline Damsky for providing the CS-1 cell line. We also thank Jeffery Edwards and Pedro Mauro for technical assistance. Finally, we thank Dr. Ellis Golub and Gerald Harrison for assistance with the Ca2+ measurements and data analysis and Dr. Meenhard Herlyn for the melanoma cell line (W-164) and for useful suggestions.

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