Mammary epithelial cell-cycle progression via the alpha(2)beta(1) integrin: unique and synergistic roles of the alpha(2) cytoplasmic domain.

The alpha(2)beta(1) integrin supports cell-cycle progression of mammary epithelial cells adherent to type I collagen matrices. Integrin collagen receptors containing the alpha(2) cytoplasmic domain stimulated expression of cyclin E and cyclin-dependent kinase (cdk)2, resulting in cyclin E/cdk2 activation in the absence of growth factors other than insulin. Integrin collagen receptors in which the alpha(2) cytoplasmic domain was replaced by the alpha(1) cytoplasmic domain or an alpha(2) subunit cytoplasmic domain truncated after the GFFKR sequence failed to stimulate cyclin E/cdk2 activation or entry into S phase in the absence of growth factors. Although overexpression of cyclins D or E or cdk2 in cells expressing the integrin collagen receptor with the alpha(1)-integrin cytoplasmic domain did not restore G(1) progression when mammary epithelial cells adhered to type I collagen, co-expression of cyclin E and cdk2 did rescue the ability of the transfectants to enter S phase. Activation of cyclin E/cdk2 complex by mammary epithelial cells required synergy between adhesion mediated by an integrin collagen receptor containing the alpha(2)-integrin subunit cytoplasmic domain and the insulin receptor.

Specific residues within the alpha 2 integrin subunit cytoplasmic domain regulate migration and cell cycle progression via distinct MAPK pathways.

The alpha(2) integrin subunit cytoplasmic domain is necessary for epidermal growth factor (EGF)-stimulated chemotactic migration and insulin-dependent entry into S-phase of mammary epithelial cells adherent to type I collagen. Truncation mutants revealed that the seven amino acids, KYEKMTK, in addition to the GFFKR motif were sufficient for these functions. Mutation of tyrosine 1134 to alanine inhibited the ability of the cells to phosphorylate p38 MAPK and to migrate in response to EGF but had only a modest effect on the ability of the cells to induce sustained phosphorylation of the ERK MAPK, to up-regulate cyclin E and cdk2 expression, and to enter S-phase when adherent to type I collagen. Conversely, mutation of the lysine 1136 inhibited the ability of the cells to increase cyclin E and cdk2 expression, to maintain long term phosphorylation of the ERK MAPK, and to enter S-phase but had no effect on the ability of the cells to phosphorylate the p38 MAPK or to migrate on type I collagen in response to EGF. Methionine 1137 was essential for both migration and entry into S-phase. Thus, distinctly different structural elements of the alpha(2) integrin cytoplasmic domain are required to engage the signaling pathways leading to cell migration or cell cycle progression.

Structural analysis of the alpha(2) integrin I domain/procollagenase-1 (matrix metalloproteinase-1) interaction.

Previous studies have established that ligation of keratinocyte alpha(2)beta(1) integrin by type I collagen induces expression of matrix metalloproteinase-1 (MMP-1) and that MMP-1 activity is required for the alpha(2)beta(1) integrin-dependent migration of primary keratinocytes across collagenous matrices. We now present evidence that MMP-1 binds the alpha(2)beta(1) integrin via the I domain of the alpha(2) integrin subunit. Using an enzyme-linked immunosorbent assay with purified human MMP-1 and recombinant alpha(2) integrin I domain, we showed that the alpha(2) integrin I domain specifically bound in a divalent cation-dependent manner to both the pro and active forms of MMP-1, but not to MMP-3 or MMP-13. Although both the I domain and MMP-1 bind divalent cations, MMP-1 bound, in a divalent cation-dependent manner, to alpha(2) integrin I domains containing metal ion-dependent adhesion sites motif mutations that prevent divalent cation binding to the I domain, demonstrating that the metal ion dependence is a function of MMP-1. Using a series of MMP-1-MMP-3 and MMP-1-MMP-13 chimeras, we determined that both the linker domain and the hemopexin-like domain of MMP-1 were required for optimal binding to the I domain. The alpha(2) integrin/MMP-1 interaction described here extends an emerging paradigm in matrix biology involving anchoring of proteinases to the cell surface to regulate their biological activities.

Pro-collagenase-1 (matrix metalloproteinase-1) binds the alpha(2)beta(1) integrin upon release from keratinocytes migrating on type I collagen.

In injured skin, collagenase-1 (matrix metalloproteinase-1 (MMP-1)) is induced in migrating keratinocytes. This site-specific expression is regulated by binding of the alpha(2)beta(1) integrin with dermal type I collagen, and the catalytic activity of MMP-1 is required for keratinocyte migration. Because of this functional association among substrate/ligand, receptor, and proteinase, we assessed whether the integrin also directs the compartmentalization of MMP-1 to its matrix target. Indeed, pro-MMP-1 co-localized to sites of alpha(2)beta(1) contacts in migrating keratinocytes. Furthermore, pro-MMP-1 co-immunoprecipitated with alpha(2)beta(1) from keratinocytes, and alpha(2)beta(1) co-immunoprecipitated with pro-MMP-1. No other MMPs bound alpha(2)beta(1), and no other integrins interacted with MMP-1. Pro-MMP-1 also provided a substrate for alpha(2)beta(1)-dependent adhesion of platelets. Complex formation on keratinocytes was most efficient on native type I collagen and reduced or ablated on denatured or cleaved collagen. Competition studies suggested that the alpha(2) I domain interacts with the linker and hemopexin domains of pro-MMP-1, not with the pro-domain. These data indicate that the interaction of pro-MMP-1 with alpha(2)beta(1) confines this proteinase to points of cell contact with collagen and that the ternary complex of integrin, enzyme, and substrate function together to drive and regulate keratinocyte migration.

Echoviruses 1 and 8 are closely related genetically, and bind to similar determinants within the VLA-2 I domain.

Echoviruses (EV) 1 and 8 were originally considered to be distinct serotypes, but more recently have been considered strains of the same virus. In experiments with chimeric recombinant fusion proteins, both viruses bound to the I domain of the integrin VLA-2, and both required the same receptor residues for attachment. A full-length, infectious cDNA clone encoding EV1 was obtained; its nucleotide sequence was determined, as were the sequences encoding the EV8 capsid. EV1 and 8 show 94% amino acid identity within the capsid region and are more similar to each other than to any other human picornavirus.

alpha 2 integrin subunit cytoplasmic domain-dependent cellular migration requires p38 MAPK.

The alpha(2) integrin subunit cytoplasmic domain uniquely supported epidermal growth factor (EGF)-stimulated migration on type I collagen. p38 MAP kinase- and phosphatidylinositol 3-kinase-specific inhibitors, but not a MEK-specific inhibitor, eliminated EGF-stimulated and unstimulated alpha(2)-cytoplasmic domain-dependent migration. Following adhesion to collagenous matrices, cells expressing the full-length alpha(2) integrin subunit, but not cells expressing a chimeric alpha(2) integrin subunit in which the alpha(2)-cytoplasmic domain was replaced by the cytoplasmic domain of the alpha(1)-subunit, exhibited sustained and robust phosphorylation of p38 MAP kinase. Expression of dominant negative p38 MAP kinase inhibited alpha(2)-cytoplasmic domain-dependent, EGF-stimulated migration as well as unstimulated migration on collagen. Expression of constitutively active Rac1(Val-12) augmented p38 MAP kinase activation and alpha(2)-cytoplasmic domain-dependent migration. It also rescued the ability of cells expressing the alpha(1)-cytoplasmic domain to activate p38 MAPK and to migrate. These results suggest that the alpha(2) integrin cytoplasmic domain uniquely stimulates the p38 MAP kinase pathway that is required for unstimulated and EGF-stimulated migration on type I collagen.

Extracellular FGF-1 inhibits cytoskeletal organization and promotes fibroblast motility.

Previous efforts from this laboratory have established that acidic fibroblast growth factor (FGF-1), either added exogenously or secreted as a biologically active protein, induces a transformed phenotype in primary murine fibroblasts. Experimental studies described here demonstrate that constitutive exposure to extracellular FGF-I results in reduced cell attachment to multiple ligands, inhibition of cytoskeletal organization, and reduced collagen contraction, despite no detectable change in integrin cell surface expression. In addition, FGF-1-transduced fibroblasts demonstrated a > 10-fold increase in migration, an observation correlated with increased tyrosine phosphorylation of p125FAK and p130CAS. Collectively, these results suggest that FGF-1-induced fibroblast transformation includes the involvement of specific FGF receptor-mediated signal transduction cascades targeted to cytoskeletal and focal adhesion structures.

Decorin binds near the C terminus of type I collagen.

Decorin belongs to a family of small leucine-rich proteoglycans that are directly involved in the control of matrix organization and cell growth. Genetic evidence indicates that decorin is required for the proper assembly of collagenous matrices. Here, we sought to establish the precise binding site of decorin on type I collagen. Using rotary shadowing electron microscopy and photoaffinity labeling, we mapped the binding site of decorin protein core to a narrow region near the C terminus of type I collagen. This region is located within the cyanogen bromide peptide fragment alpha1(I) CB6 and is approximately 25 nm from the C terminus, in a zone that coincides with the c(1) band of the collagen fibril d-period. This location is very close to one of the major intermolecular cross-linking sites of collagen heterotrimers. Thus, decorin protein core possesses a unique binding specificity that could potentially regulate collagen fibril stability.

Role of the alpha1 and alpha2 integrin cytoplasmic domains in cell morphology, motility and responsiveness to stimulation by the protein kinase C pathway.

The alpha1beta1 and alpha2beta1 integrins, extracellular matrix receptors for collagens and/or laminins, have similarities in structure and ligand binding. Recent studies suggest that the two receptors mediate distinct post-ligand binding events and are not simply redundant receptors. To discern the mechanisms by which the two receptors differ, we focused on the roles of the cytoplasmic domains of the alpha subunits. We expressed either full-length alpha1 integrin subunit cDNA (X1C1), full-length alpha2 integrin subunit cDNA (X2C2), chimeric cDNA composed of the extracellular and transmembrane domains of alpha2 subunit and the cytoplasmic domain of alpha1 (X2C1), chimeric cDNA composed of the extracellular and transmembrane domains of alpha1 subunit and the cytoplasmic domain of alpha2 (X1C2), alpha1 cDNA truncated after the GFFKR sequence (X1C0) or alpha2 cDNA truncated after the GFFKR sequence (X2C0) in K562 cells. Although the cytoplasmic domains of the alpha1 and alpha2 subunits were not required for adhesion, the extent of adhesion at low substrate density was enhanced by the presence of either the alpha1 or alpha2 cytoplasmic tail. Spreading was also influenced by the presence of an alpha subunit cytoplasmic tail. Activation of the protein kinase C pathway with phorbol dibutyrate-stimulated motility that was dependent upon the presence of the alpha2 cytoplasmic tail. Both the phosphatidylinosotide-3-OH kinase and the mitogen-activated protein kinase pathways were required for phorbol-activated, alpha2-cytoplasmic tail-dependent migration.

Determinants of ligand binding specificity of the alpha(1)beta(1) and alpha(2)beta(1) integrins.

The alpha(1)beta(1) and alpha(2)beta(1) integrins are cell surface collagen receptors. Cells expressing the alpha(1)beta(1) integrin preferentially adhere to collagen IV, whereas cells expressing the alpha(2)beta(1) integrin preferentially adhere to collagen I. Recombinant alpha(1) and alpha(2) integrin I domains exhibit the same collagen type preferences as the intact integrins. In addition, the alpha(2) integrin I domain binds echovirus 1; the alpha(1) I domain does not. To identify the structural components of the I domains responsible for the varying ligand specificities, we have engineered several alpha(1)/alpha(2) integrin I domain chimeras and evaluated their virus and collagen binding activities. Initially, large secondary structural components of the alpha(2) I domain were replaced with corresponding regions of the alpha(1) I domain. Following analysis in echovirus 1 and collagen binding assays, chimeras with successively smaller regions of alpha(1) I were constructed and analyzed. The chimeras were analyzed by ELISA with several different alpha(2) integrin monoclonal antibodies to assess their proper folding. Three different regions of the alpha(1) I domain, when present in the alpha(2) I domain, conferred enhanced collagen IV binding activity upon the alpha(2) I domain. These include the alpha3 and alpha5 helices and a portion of the alpha6 helix. Echovirus 1 binding was lost in a chimera containing the alphaC-alpha6 loop; higher resolution mapping identified Asn(289) as playing a critical role in echovirus 1 binding. Asn(289) had not been implicated in previous echovirus 1 binding studies. Taken together, these data reveal the existence of multiple determinants of ligand binding specificities within the alpha(1) and alpha(2) integrin I domains.

Collagen receptor control of epithelial morphogenesis and cell cycle progression.

To define the unique contributions of the alpha subunit cytoplasmic tails of the alpha(1)beta(1) and alpha(2)beta(1) integrin to epithelial differentiation and branching morphogenesis, a variant NMuMG cell line lacking alpha(1)beta(1) and alpha(2)beta(1) integrin expression was stably transfected with the full-length alpha(2) integrin subunit cDNA (X2C2), chimeric cDNA consisting of the extracellular and transmembrane domains of the alpha(2) subunit and the cytoplasmic domain of the alpha(1) subunit (X2C1), or alpha(2) cDNA truncated after the GFFKR sequence (X2C0). The X2C2 and X2C1 transfectants effectively adhered, spread, and formed focal adhesion complexes on type I collagen matrices. The X2C0 transfectants were less adherent to low concentrations of type I collagen, spread less well, and formed poorly defined focal adhesion complexes in comparison to the X2C2 and X2C1 transfectants. The X2C2 and X2C1 transfectants but not the X2C0 transfectants proliferated on collagen substrates. Only the X2C2 transfectants developed elongate branches and tubules in three-dimensional collagen gels and migrated on type I collagen. These findings suggest a unique role for the alpha(2) integrin cytoplasmic domain in postligand binding events and cooperative interactions with growth factors that mediate epithelial differentiation and branching morphogenesis. Either intact alpha(1) or alpha(2) integrin subunit cytoplasmic domain can promote cell cycle progression.

Integrins and the kidney: biology and pathobiology.

Integrins represent a superfamily of cell surface molecules that are important mediators of cell-extracellular matrix interactions. Of the many known integrin subunit combinations, only a few (alpha 1 beta 1, alpha 2 beta 1, alpha 3 beta 1, alpha 6 beta 1, alpha 8 beta 1 and alpha v beta 3) appear to play significant roles in renal development and function. The current understanding of these roles is reviewed. Potential therapeutic benefits from the alteration of integrin function by arginine-glycine-aspartic acid peptides in renal ischemic injury have been suggested. Reduced tubular obstruction is a potential mechanism, however other mechanisms remain to be explored. Finally, recent studies suggest a mechanism whereby abnormal interactions between integrins and non-specifically glycosylated glomerular basement membrane components could be involved in the pathogenesis of diabetic nephropathy. The elucidation of other potential pathophysiological roles for integrins in renal disease has just begun.

Binding of the alpha 2 integrin I domain to extracellular matrix ligands: structural and mechanistic differences between collagen and laminin binding.

The alpha 2 beta 1 integrin functions as a cell surface receptor for collagen on some cells and as both a collagen and laminin receptor on a more restricted subset of cell types including endothelial and epithelial cells. The alpha 2 integrin subunit I domain binds collagen in a divalent cation-dependent manner. In contrast, I domain binding to laminin occurs via both divalent cation-dependent and -independent mechanisms. Saturable binding was observed in the presence of either Mn2+ or EDTA, although the extent of binding in Mn2+ was twice that observed in EDTA. Half-maximal binding occurred at about 22 nM I domain in either case. Whereas laminin binding was significantly enhanced by Mn2+, with half-maximal binding occurring at 1.9 mM Mn2+, Mg2+ was much less effective. Deletion of the N-terminal 35 residues of the I domain, including the DXSXS portion of the MIDAS motif, caused a significant diminution of laminin binding activity. Laminin binding by the I domain was significantly inhibited by the alpha 2 beta 1 function-blocking antibody 6F1 in the presence of either EDTA or Mn2+. The non-function-blocking antibody 12F1 had no effect. In contrast to the binding of the alpha 2 integrin I domain to collagen, the laminin binding activity of the I domain was not enhanced by the addition of the first EF hand motif of the integrin.

Ligand binding results in divalent cation displacement from the alpha 2 beta 1 integrin I domain: evidence from terbium luminescence spectroscopy.

The alpha 2 beta 1 integrin serves as a cell surface collagen or collagen/laminin receptor. Binding of the integrin to its ligands is largely mediated by the alpha 2 subunit I domain and requires the presence of divalent cations. Terbium ion (Tb3+), a fluorescent trivalent cation that often binds divalent cation-binding sites on proteins, supported binding of the I domain to collagen with half-maximal binding occurring at 5.2 +/- 1.7 microM Tb3+. By fluorescence resonance energy transfer spectroscopy, Tb3+ showed specific and saturable binding to the recombinant I domain with a Kd of 27 +/- 4 microM. Although both Mg2+ and Mn2+ were capable of quenching Tb3+ fluorescence, Mn2+ was much more effective than Mg2+. The alpha 2 beta 1 integrin also binds the pro-alpha 1(I) collagen carboxyl-terminal propeptide in a Mg2+-dependent manner via the I domain. Recombinant propeptide was used to examine the effect of ligand on the Tb3+ binding properties of the alpha 2 integrin I domain. As propeptide bound to the I domain, Tb3+ fluorescence progressively diminished suggesting that as ligand binds to the I domain, either Tb3+ is displaced or its fluorescence is quenched. Consistent with the former possibility, little dissociation of collagen-bound I domain occurred upon the addition of EDTA and subsequent incubation. These data support a model in which (1) the divalent cation is required for initial ligand-binding activity of the I domain and (2) ligand binding results in subsequent metal ion displacement to generate a metal-free I domain-ligand complex.

Alpha2beta1 integrin recognition of the carboxyl-terminal propeptide of type I procollagen: integrin recognition and feed-back regulation of matrix biosynthesis are mediated by distinct sequences.

It has recently been established that the carboxyl-terminal propeptide of type I collagen exert a feedback regulatory effect on extracellular matrix biosynthesis and that the propeptide bind to the alpha2beta1 integrin. This raises the intriguing hypothesis that the regulatory propeptide sequences exert their effects as a consequence of binding to the integrin. We show that recombinant alpha1(I) collagen chain C-terminal propeptide contains a binding site for the intact alpha2beta1 integrin and for a recombinant alpha2 integrin I domain, but not for the alpha1beta1 integrin, a structurally and functionally related collagen/laminin receptor. Additional studies employing a series of recombinant N-terminal and C-terminal deletion mutants, internal fragments of the propeptide, synthetic peptides, recombinant alpha2 integrin I domain and inhibitory monoclonal antibodies established that the previously identified sequences within the alpha1(I) C-terminal propeptide that mediate regulation of matrix biosynthesis are neither necessary nor sufficient for alpha2beta1 integrin binding. In contrast, the integrin recognition site is composed of a conformationally complex determinant located within a structurally distinct 115 amino acid region of the propeptide.

Ligand recognition by the I domain-containing integrins.

Seven of the integrin alpha subunits described to date, alpha 1, alpha 2, alpha L, alpha X, alpha d, alpha M and alpha E, contain a highly conserved I (or A) domain of approximately 200 amino acid residues inserted near the amino-terminus of the subunit. As the result of a variety of independent experimental approaches, a large body of data has recently accumulated that indicates that the I domains are independent, autonomously folding domains capable of directly binding ligands that play a necessary and important role in ligand binding by the intact integrins. Recent crystallographic studies have elucidated the structures of recombinant alpha M and alpha L I domains and also delineated a novel divalent cation-binding motif within the I domains (metal ion-dependent adhesion site, MIDAS) that appears to mediate the divalent cation binding of the I domains and the I domain-containing integrins to their ligands.

Downstream events in mammary gland morphogenesis mediated by reexpression of the alpha2beta1 integrin: the role of the alpha6 and beta4 integrin subunits.

Our previous studies demonstrated that reexpression of the alpha2beta1 integrin by a poorly differentiated breast carcinoma cell line, Mm5MT, resulted in dramatic reversion of a malignant phenotype to a differentiated epithelial phenotype. We hypothesized that reexpression of the alpha2beta1 integrin may regulate expression of other genes, the expression of which contributed to the dramatic phenotypic change. We now show that reexpression of the alpha2beta1 integrin results in up-regulation of both the alpha6 and beta4 integrin subunits but no change in the alpha1, alpha3, alpha5, or beta1 integrin subunits or E-cadherin. To further investigate the role of the alpha6 and beta4 integrin subunits in mediating the phenotypic changes elicited by reexpression of the alpha2beta1 integrin, the alpha6 or beta4 integrin subunit was expressed in our Mm5MT model. Expression of either subunit increased adhesion to laminin-1. Although adhesion to collagen was unaltered, contraction of three-dimensional collagen gels was reduced. Expression of either the alpha6 or beta4 integrin subunit also restored some aspects of a less malignant phenotype, including the acquisition of contact inhibition and diminution of anchorage-dependent and anchorage-independent growth rates. The alpha6 and beta4 transfectants formed three-dimensional organized structures when grown in gels of reconstituted basement membrane but did not form the highly branched, duct-like structures formed by the alpha2 transfectants. In contrast to the reduced invasiveness of the alpha2 transfectants, the alpha6 and beta4 transfectants retained an invasive phenotype. These results suggest that expression of the alpha6beta4 integrin contributes to some but not all of the phenotypic changes elicited by reexpression of the alpha2 integrin subunit and modulates the function of other integrins on these cells. Using our Mm5MT model, we are defining the cascade of integrin expression required for maintenance of the differentiated mammary epithelial cell phenotype.

A three-dimensional collagen lattice activates NF-kappaB in human fibroblasts: role in integrin alpha2 gene expression and tissue remodeling.

Normal adult human dermal fibroblasts grown in a three-dimensional collagen lattice increase mRNA level of collagen receptor integrin subunit alpha2 (Xu, J., and R.A.F. Clark. 1996. J. Cell Biol. 132:239- 249.) and DNA binding activity of a nuclear transcription factor, NF-kappaB (Xu, J., and R.A.F. Clark. 1997. J. Cell Biol. 136:473-483.). Here we present evidence that the collagen lattice induced the nuclear translocation of p50, one member of NF-kappaB family, and the degradation of an NF-kappaB inhibitor protein, IkappaB-alpha. The inhibition of NF-kappaB activity by SN50, a peptide inhibitor targeted at nuclear translocation of NF-kappaB, significantly reduced the induction of integrin alpha2 mRNA and protein by the collagen lattice. A region located between -549 and -351 bp in the promoter of integrin alpha2 gene conferred the inducibility by three-dimensional collagen lattice. The presence of either SN50 or IkappaB-alpha32, 36, a stable mutant of IkappaB-alpha, abrogated this inducibility, indicating that the activation of integrin alpha2 gene expression was possibly mediated by NF-kappaB through this region. Although there were three DNA-protein binding complexes forming in this region that are sensitive to the inhibition of NF-kappaB nuclear translocation, NF-kappaB was not directly present in the binding complexes. Therefore, an indirect regulatory mechanism by NF-kappaB in integrin alpha2 gene expression induced by three-dimensional collagen lattice is suggested. The involvement of NF-kappaB in reorganization and contraction of three-dimensional collagen lattice, a process that requires the presence of abundant integrin alpha2beta1, was also examined. The inhibition of NF-kappaB activity by SN50 greatly blocked the contraction, suggesting its critical role in not only the induction of integrin alpha2 gene expression by three-dimensional collagen lattice, but also alpha2beta1-mediated tissue-remodeling process.