Wound healing in the skin requires a compromise between adhesion and migration. Both processes include modulation of the cytoskeleton, cell-surface receptors, and receptor ligands., In this issue, Kopecki et al. demonstrate that overexpression of Flii, an actin-remodeling protein, impedes wound healing but inhibits hemidesmosome formation. In contrast, Flii deficiency results in enhanced wound healing while promoting hemidesmosome assembly. We discuss potential mechanisms that could explain how this unique gelsolin family member might regulate both stable keratinocyte adhesion and motility.
α6β4 integrin, a component of hemidesmosomes, also plays a role in keratinocyte migration via signaling through Rac1 to the actin-severing protein cofilin. Here, we tested the hypothesis that the β4 integrin-associated plakin protein, bullous pemphigoid antigen 1e (BPAG1e) functions as a scaffold for Rac1/cofilin signal transduction. We generated keratinocyte lines exhibiting a stable knockdown in BPAG1e expression. Knockdown of BPAG1e does not affect expression levels of other hemidesmosomal proteins, nor the amount of β4 integrin expressed at the cell surface. However, the amount of Rac1 associating with β4 integrin and the activity of both Rac1 and cofilin are significantly lower in BPAG1e-deficient cells compared with wild-type keratinocytes. In addition, keratinocytes deficient in BPAG1e exhibit loss of front-to-rear polarity and display aberrant motility. These defects are rescued by inducing expression of constitutively active Rac1 or active cofilin. These data indicate that the BPAG1e is required for efficient regulation of keratinocyte polarity and migration by determining the activation of Rac1.
Mouse keratinocytes migrate significantly slower than their human counterparts in vitro on uncoated surfaces. We tested the hypothesis that this is a consequence of differences in the extracellular matrix (ECM) that cells deposit. In support of this, human keratinocyte motility was dramatically reduced when plated onto the ECM of mouse skin cells whereas the latter cells migrated faster when plated onto human keratinocyte ECM. The ECM of mouse and human keratinocytes contained similar levels of the α3 laminin subunit of laminin-332. However, mouse skin cells expressed significantly more fibronectin (FN) than human cells. To assess whether FN is a motility regulator, we utilized siRNA to reduce expression of FN in mouse keratinocytes. The treated mouse keratinocytes moved significantly more rapidly than wild-type mouse skin cells. Moreover, the FN depleted mouse cell ECM supported increased migration of both mouse and human keratinocytes. Furthermore, the motility of human keratinocytes was slowed when plated onto FN-coated substrates or human keratinocyte ECM supplemented with FN in a dose dependent manner. Consistent with these findings, the ECM of α3 integrin-null keratinocytes, which also migrated faster than wild-type cells, was FN deficient. Our results provide evidence that FN is a brake to skin cell migration supported by laminin-332-rich matrices.
Transdominant inhibition of integrins or integrin-integrin crosstalk is an important regulator of integrin ligand binding and subsequent signaling events that control a variety of cell functions in many tissues. Here we discuss examples of integrin crosstalk and detail our current understanding of the molecular mechanisms that are involved in this receptor phenomenon. The cytoskeleton associated protein talin is a key regulator of integrin crosstalk. We describe how the interaction of talin and the cytoplasmic tail of β integrin is controlled and how competitive inhibitors of this binding play a role in integrin crosstalk. We conclude with a discussion of how integrin crosstalk impacts the interpretation of integrin inhibitor and knockdown studies in both the laboratory and clinical setting.
Matrix adhesion; receptors; cytoskeleton; signaling; adaptor proteins; phosphorylation
Basement membrane plays important roles in hair growth. We characterized changes in laminin isoform expression during hair cycling. At the mRNA level, laminin-511 (10) expression underwent a steady increase during anagen stages. In contrast, laminin-332 (5) expression was initially upregulated in outer root sheath (ORS) keratinocytes at anagen II and then transiently downregulated. Laminin-332 significantly increased coincident with the signal in inner root sheath and hair matrix cells after anagen IV. Levels of laminin-332 proteins were also upregulated at late anagen I–III but dropped after anagen IV. This decrease coincided with increased levels of mRNA encoding the two proteases, membrane type 1 metalloproteinase and bone morphogenetic protein 1, involved in laminin-332 processing. Immunohistochemistry demonstrated that laminin-332 and α6β4 integrin were well colocalized, but their signals were remarkably decreased in the lower half of follicles after anagen VI. Consistent with these data, ultrastructurally mature hemidesmosomes were seen in ORS keratinocytes at anagen II, whereas at anagen VI, only fragmental hemidesmosomes were present. In hair follicle culture, laminin-511 (10)/521 (11)-rich human placental laminin enhanced hair growth, whereas recombinant laminin-332 antagonized hair growth induced by laminin-511. Our results indicate a positive role for laminin-511 and a negative role for laminin-332 on hair growth.
laminin; hemidesmosome; integrin; protease; hair cycle
For many years, extracellular matrix (ECM) was considered to function as a tissue support and filler. However, we now know that ECM proteins control many cellular events through their interaction with cell-surface receptors and cytoplasmic signaling pathways. For example, they regulate cell proliferation, cell division, cell adhesion, cell migration, and apoptosis. We focus in this review on a laminin isoform, laminin-332 (formerly termed laminin-5), a major component of the basement membrane (BM) of skin and other epithelial tissues. It is composed of 3 subunits (α3, β3, and γ2) and interacts with at least two integrin receptors expressed by epithelial cells (α3β1 and α6β4 integrin). Mutations in either laminin-332 or integrin α6β4 result in junctional epidermolysis bullosa, a blistering skin disease, while targeting of laminin-332 by autoantibodies in cicatricial pemphigoid leads to dysadhesion of epithelial cells from their underlying connective tissue. Abnormal expression of laminin-332 and its integrin receptors is also a hallmark of certain tumor types and is believed to promote invasion of colon, breast and skin cancer cells. Moreover, there is emerging evidence that laminin-332 and its protease degradation products are not only found at the leading front of several tumors but also likely induce and/or promote tumor cell migration. Thus, in this review, we focus specifically on the role of laminin-332 and its integrin receptors in adhesion, proliferation, and migration/invasion of cancer cells. Finally, we discuss strategies for the development of laminin-332-based antagonists for the treatment of malignant tumors.
Integrin; laminin; cancer; basement membrane; proteolysis; cell signaling; cell adhesion; gene expression
The protein bullous pemphigoid antigen-2 (BPAG2/BP180/collagen type XVII) plays a key role in attachment of basal keratinocytes to epidermal basement membrane. The binding of BP180 with either integrin α6, integrin β4, or bullous pemphigoid antigen-1 (BPAG1/ BP230) is critical for this attachment in skin. The protein 14-3-3 σ, also known as stratifin and a marker for epithelial cells, is a member of a highly conserved small acidic 14-3-3 protein family naturally found in all eukaryotic cells. Here, we have used a 14-3-3σ GST pull-down screening assay and showed that sigma (σ) isoform of the 14-3-3 protein family interacts with the cytoplasmic N-terminal domain of BP180. Analysis of a series of truncated or deleted 14-3-3σ revealed that only intact 14-3-3σ molecule, but not any of its fragments can interact with BP180. This finding suggests that conformation and possible dimerization of 14-3-3 σ is essential for this interaction. Further, a BP180 co-immunoprecipitation (IP) and its reverse IP assays were conducted and the results confirmed that 14-3-3 σ interacts with cytoplasmic domain, but not ecto-domain of the BP180. In conclusion, the finding of this study provides evidence that 14-3-3σ isoform interacts with BP180 which is a major component of hemidesmosome involved in the attachment of epidermis to the basement membrane in skin. However, the significance of this interaction in hemidesmosome formation and/or attachment needs to be explored.
The tetraspanin CD151 forms complexes in epithelial cell membranes with laminin-binding integrins α6 β4, α3 β1, and α6 β1, and modifies integrin-mediated cell migration in vitro. We demonstrate in this study that CD151 expression is upregulated in a distinct temporal and spatial pattern during wound healing, particularly in the migrating epidermal tongue at the wound edge, suggesting a role for CD151 in keratinocyte migration. We show that healing is significantly impaired in CD151-null mice, with wounds gaping wider at 7 days post-injury. The rate of re-epithelialization of the CD151-null wounds is adversely affected, with significantly less wound area being covered by migrating epidermal cells. Our studies reveal that although laminin levels are similar in wild-type and CD151-null wounds, the organization of the laminin in the basement membrane is impaired. Furthermore, upregulation of α6 and β4 integrin expression is adversely affected in CD151-null mice wounds. In contrast, we find no significant effect of CD151 gene knockout on α3 and β1 integrin expression in wound repair. We suggest that mice lacking the CD151 gene are defective in wound healing, primarily owing to impairment of the re-epithelialization process. This may be due to defective basement membrane formation and epithelial cell adhesion and migration.
Defining the pathways required for keratinocyte cell migration is important for understanding mechanisms of wound healing and tumor cell metastasis. We have recently identified an α6β4 integrin-Rac1 signaling pathway via which the phosphatase Slingshot (SSH) activates/dephosphorylates cofilin, thereby determining keratinocyte migration behavior. Here, we assayed the role of 14-3-3 isoforms in regulating the activity of SSH1. Using amino or carboxy terminal domains of 14-3-3ζ we demonstrate that in keratinocytes 14-3-3ζ/τ heterodimers bind SSH1, in the absence of Rac1 signaling. This interaction leads to an inhibition of SSH1 activity, as measured by an increase in phosphorylated cofilin levels. Overexpression of the carboxy terminal domain of 14-3-3ζ acts as a dominant negative and inhibits the interaction between 14-3-3τ and SSH1. These results implicate 14-3-3ζ/τ heterodimers as key regulators of SSH1 activity in keratinocytes and suggest they play a role in cytoskeleton remodeling during cell migration.
Slingshot; 14-3-3; migration; keratinocytes
Epidermal cells adhere to the basement membrane zone through cell–matrix junctions termed hemidesmosomes. During wound healing, hemidesmosomes are disassembled to allow keratinocytes to move over wound sites. Such movement is mediated by both hemidesmosome protein complexes (HPCs) and focal contacts (FCs). In this study, we analyzed the interaction between HPCs and FCs in live HaCat cells expressing yellow fluorescent protein (YFP)-tagged β4 integrin and cyan fluorescent protein (CFP)-tagged α-actinin as markers of HPCs and FCs, respectively. In HaCat cells migrating to repopulate wounds, FC proteins cluster rapidly in the direction of the wound. HPC assembly then follows and the newly formed HPCs occupy sites vacated by the disassembled FCs. HPC dynamics are dramatically reduced, and HaCat cells cease migration upon treatment with reagents that affect FC integrity/function. Upon treatment with reagents that destabilize HPCs, the dynamics of FCs in HaCat cells at the edges of wounds are enhanced, although FC assembly is irregular and the migration of the cells is aberrant. We also show that the complex interaction between hemidesmosomes and FCs in keratinocytes is myosin dependent and requires energy. In summary, we suggest that HPCs and FCs dynamics are tightly co-regulated in keratinocytes undergoing migration during wound healing.
Chemotherapy-induced alopecia (CIA) has a devastating cosmetic effect, especially in the young. Recent data indicate that two major basement membrane components (laminin-332 and -511) of the skin have opposing effects on hair growth.
In this study, we examined the role and localization of laminin-332 and -511 in CIA.
We examined the expression of laminin-332 and -511 during the dystrophic catagen form of CIA induced in C57BL/6 mice by cyclophosphamide (CYP) treatment.
Our data indicate that both laminin-332 and its receptor α6β4 integrin are up-regulated (both quantitatively and spatially) after mid to late dystrophic catagen around the outer root sheath (ORS) in the lower third of hair follicles in CIA. This up-regulation also occurs at the transcriptional level. In contrast, laminin-511 is down-regulated after mid dystrophic catagen at the protein level, with transcriptional inactivation of laminin-511 occurring transiently at the early dystrophic catagen stage in both epidermal and ORS keratinocytes. Laminin-511 expression correlates with expression of α3 integrin in CIA and we also demonstrate that laminin-511 can up-regulate the activity of the α3 integrin promoter in cultured keratinocytes. Injection of a laminin-511 rich protein extract, but not recombinant laminin-332, in the back skin of mice delays hair loss in CYP-induced CIA.
We propose that abrupt hair loss in CIA is, at least in part, caused by down-regulation of laminin-511 and up-regulation of laminin-332 at the transcriptional and translational levels.
A549 cells; Chemotherapy; Extracellular matrix; Hemidesmosome; Integrin; Outer root sheath cells
Much effort has been expended on analyzing how microfilament and microtubule cytoskeletons dictate the interaction of cells with matrix at adhesive sites called focal adhesions (FAs). However, vimentin intermediate filaments (IFs) also associate with the cell surface at FAs in endothelial cells. Here, we show that IF recruitment to FAs in endothelial cells requires β3 integrin, plectin and the microtubule cytoskeleton, and is dependent on microtubule motors. In CHO cells, which lack β3 integrin but contain vimentin, IFs appear to be collapsed around the nucleus, whereas in CHO cells expressing β3 integrin (CHOwtβ3), vimentin IFs extend to FAs at the cell periphery. This recruitment is regulated by tyrosine residues in the β3 integrin cytoplasmic tail. Moreover, CHOwtβ3 cells exhibit significantly greater adhesive strength than CHO or CHO cells expressing mutated β3 integrin proteins. These differences require an intact vimentin network. Therefore, vimentin IF recruitment to the cell surface is tightly regulated and modulates the strength of adhesion of cells to their substrate.
Intermediate filament; Integrin; Adhesion
Whether α6β4 integrin regulates migration remains controversial. β4 integrin-deficient (JEB) keratinocytes display aberrant migration in that they move in circles, a behavior that mirrors the circular arrays of laminin (LM)-332 in their matrix. In contrast, wild-type keratinocytes and JEB keratinocytes, induced to express β4 integrin, assemble laminin-332 in linear tracks over which they migrate. Moreover, laminin-332-dependent migration of JEB keratinocytes along linear tracks is restored when cells are plated on wild-type keratinocyte matrix, whereas wild-type keratinocytes show rotation over circular arrays of laminn-332 in JEB keratinocyte matrix. The activities of Rac1 and the actin cytoskeleton-severing protein cofilin are low in JEB keratinocytes compared with wild-type cells but are rescued following expression of wild-type β4 integrin in JEB cells. Additionally, in wild-type keratinocytes Rac1 is complexed with α6β4 integrin. Moreover, Rac1 or cofilin inactivation induces wild-type keratinocytes to move in circles over rings of laminin-332 in their matrix. Together these data indicate that laminin-332 matrix organization is determined by the α6β4 integrin/actin cytoskeleton via Rac1/cofilin signaling. Furthermore, our results imply that the organizational state of laminin-332 is a key determinant of the motility behavior of keratinocytes, an essential element of skin wound healing and the successful invasion of epidermal-derived tumor cells.
Mechanical ventilation is a valuable treatment regimen for respiratory failure. However, mechanical ventilation (especially with high tidal volumes) is implicated in the initiation and/or exacerbation of lung injury. Hence, it is important to understand how the cells that line the inner surface of the lung [alveolar epithelial cells (AECs)] sense cyclic stretching. Here, we tested the hypothesis that matrix molecules, via their interaction with surface receptors, transduce mechanical signals in AECs. We first determined that rat AECs secrete an extracellular matrix (ECM) rich in anastamosing fibers composed of the α3 laminin subunit, complexed with β1 and γ1 laminin subunits (i.e. laminin-6), and perlecan by a combination of immunofluorescence microscopy and immunoblotting analyses. The fibrous network exhibits isotropic expansion when exposed to cyclic stretching (30 cycles per minute, 10% strain). Moreover, this same stretching regimen activates mitogen-activated-protein kinase (MAPK) in AECs. Stretch-induced MAPK activation is not inhibited in AECs treated with antagonists to α3 or β1 integrin. However, MAPK activation is significantly reduced in cells treated with function-inhibiting antibodies against the α3 laminin subunit and dystroglycan, and when dystroglycan is knocked down in AECs using short hairpin RNA. In summary, our results support a novel mechanism by which laminin-6, via interaction with dystroglycan, transduces a mechanical signal initiated by stretching that subsequently activates the MAPK pathway in rat AECs. These results are the first to indicate a function for laminin-6. They also provide novel insight into the role of the pericellular environment in dictating the response of epithelial cells to mechanical stimulation and have broad implications for the pathophysiology of lung injury.
Matrix adhesion; Matrix receptors; Stretching
Caspases are a conserved family of cell death proteases that cleave intracellular substrates at Asp residues to modify their function and promote apoptosis. In this report we identify the integrin β4 subunit as a novel caspase substrate using an expression cloning strategy. Together with its α6 partner, α6β4 integrin anchors epithelial cells to the basement membrane at specialized adhesive structures known as hemidesmosomes and plays a critical role in diverse epithelial cell functions including cell survival and migration. We show that integrin β4 is cleaved by caspase-3 and -7 at a conserved Asp residue (Asp1109) in vitro and in epithelial cells undergoing apoptosis, resulting in the removal of most of its cytoplasmic tail. Caspase cleavage of integrin β4 produces two products, 1) a carboxyl-terminal product that is unstable and rapidly degraded by the proteasome and 2) an amino-terminal cleavage product (amino acids 1–1109) that is unable to assemble into mature hemidesmosomes. We also demonstrate that caspase cleavage of integrin β4 sensitizes epithelial cells to apoptosis and inhibits cell migration. Taken together, we have identified a previously unrecognized proteolytic truncation of integrin β4 generated by caspases that disrupts key structural and functional properties of epithelial cells and promotes apoptosis.
Tumor cells can evade chemotherapy by acquiring resistance to apoptosis. We investigated the molecular mechanism whereby malignant and nonmalignant mammary epithelial cells become insensitive to apoptosis. We show that regardless of growth status, formation of polarized, three-dimensional structures driven by basement membrane confers protection to apoptosis in both nonmalignant and malignant mammary epithelial cells. By contrast, irrespective of their malignant status, nonpolarized structures are sensitive to induction of apoptosis. Resistance to apoptosis requires ligation of β4 integrins, which regulates tissue polarity, hemidesmosome formation, and NFκB activation. Expression of β4 integrin that lacks the hemidesmosome targeting domain interferes with tissue polarity and NFκB activation and permits apoptosis. These results indicate that integrin-induced polarity may drive tumor cell resistance to apoptosis-inducing agents via effects on NFκB.
Lung cells are exposed to cyclic stretch during normal respiration and during positive pressure mechanical ventilation administered to support gas exchange. Dystroglycan is a ubiquitously expressed matrix receptor that is required for normal basement membrane formation during embryogenesis and for maintaining the function of skeletal muscle myocytes and neurons where it links cells to matrix. We previously reported that equibiaxial stretch of primary alveolar epithelial cells activated the MAP kinase pathway ERK1/2 through a mechanism that required an interaction between dystroglycan and matrix. We determined whether this mechanism of mechanotransduction activates other signaling cascades in lung epithelium. Exposure of rat epithelial alveolar type II cells (AEC) to cyclic mechanical stretch resulted in activation of 5′ AMP-activated protein kinase (AMPK). This response was not affected by pretreatment of AEC with the ERK inhibitor PD98059 but was inhibited by knockdown in dystroglycan expression. Moreover, production of reactive oxygen species was enhanced in mechanically stimulated AEC in which dystroglycan was knocked down. This enhancement was reversed by treatment of AEC with an AMPK activator. Activation of AMPK was also observed in lung homogenates from mice after 15 minutes of noninjurious mechanical ventilation. Furthermore, knockdown of dystroglycan in the lungs of mice using an adenovirus encoding a dystroglycan shRNA prevented the stretch-induced activation of AMPK. These results suggest that exposure to cyclic stretch activates the metabolic sensing pathway AMPK in the lung epithelium and supports a novel role for dystroglycan in this mechanotransduction.
stretch; lung injury; mechanical ventilation; acute respiratory distress syndrome
The motility of keratinocytes is an essential component of wound closure and the development of epidermal tumors. In vitro, the specific motile behavior of keratinocytes is dictated by the assembly of laminin-332 tracks, a process that is dependent upon α6β4 integrin signaling to Rac1 and the actin-severing protein cofilin. Here we have analyzed how cofilin phosphorylation is regulated by phosphatases (slingshot (SSH) or chronophin (CIN)) downstream of signaling by α6β4 integrin/Rac1 in human keratinocytes. Keratinocytes express all members of the SSH family (SSH1, SSH2, and SSH3) and CIN. However, expression of phosphatase-dead versions of all three SSH proteins, but not dominant inactive CIN, results in phosphorylation/inactivation of cofilin, changes in actin cytoskeleton organization, loss of cell polarity, and assembly of aberrant arrays of laminin-332 in human keratinocytes. SSH activity is regulated by 14-3-3 protein binding, and intriguingly, 14-3-3/α6β4 integrin protein interaction is required for keratinocyte migration. We wondered whether 14-3-3 proteins function as regulators of Rac1-mediated keratinocyte migration patterns. In support of this hypothesis, inhibition of Rac1 results in an increase in 14-3-3 protein association with SSH. Thus, we propose a novel mechanism in which α6β4 integrin signaling via Rac1, 14-3-3 proteins, and SSH family members regulates cofilin activation, cell polarity, and matrix assembly, leading to specific epidermal cell migration behavior.
The laminins are a family of heterotrimeric basement membrane proteins that play roles in cellular adhesion, migration, and tissue morphogenesis. Through in silico analysis of the laminin-encoding genes, we identified a novel family of alternate splice isoforms derived from the 5′-end of the LAMA3 and LAMA5 genes. These isoforms resemble the netrins in that they contain a laminin N-terminal domain followed by a short stretch of laminin-type epidermal growth factor-like repeats. We suggest the terms LaNt (laminin N terminus) α3 and LaNt α5, for the predicted protein products of these mRNAs. RT-PCR confirmed the presence of these transcripts at the mRNA level. Moreover, they exhibit differential, tissue-specific, expression profiles. To confirm the existence of LaNt α3 protein, we generated an antibody to a unique domain within the putative polypeptide. This antibody recognizes a protein at the predicted molecular mass of 64 kDa by immunoblotting. Furthermore, immunofluorescence analyses revealed a basement membrane staining in epithelial tissue for LaNt α3 and LaNt α3 localized along the substratum-associated surface of cultured keratinocytes. We have also tested the functionality LaNt α3 through RNAi-mediated knockdown. Keratinocytes exhibiting specific knockdown of LaNt α3 displayed impaired adhesion, stress resistance, and reduced ability to close scratch wounds in vitro.
Recent analyses of collagen, elastin and fibronectin matrix assembly, organization and remodeling have been facilitated by the use of tagged proteins that can be visualized without the need for antibody labeling. Here, we report the generation of C-terminal tagged, full-length and “processed” (α3ΔLG4-5) human α3 as well as C-terminal tagged, full-length human β3 laminin subunits in adenoviral vectors. Human epidermal keratinocytes (HEKs) and human bronchial epithelial (BEP2D) cells, which assemble laminin-332-rich matrices, as well as primary rat lung alveolar type II (ATII) cells, which elaborate a fibrous network rich in laminin-311, were infected with adenovirus encoding the tagged human laminin subunits. In HEKs and BEP2D cells, tagged, full-length α3, α3ΔLG4-5 and β3 laminin subunits incorporate into arrays of matrix organized into patterns that are comparable to those observed when such cells are stained using laminin-332 subunit antibody probes. Moreover, HEKs and BEP2Ds move over these tagged, laminin-332-rich matrix arrays. We have also used the tagged β3 laminin subunit-containing matrices to demonstrate that assembled laminin-332 arrays influence laminin matrix secretion and/or assembly. In the case of rat ATII cells, although tagged α3 laminin subunits are not detected in the matrix of rat ATII cells infected with virus encoding full-length human α3 laminin protein, processed human α3 laminin subunits are incorporated into an extracellular fibrous array. We discuss how these novel laminin reagents can be used to study the organization, processing and assembly of laminin matrices and how they provide new insights into the potential functional importance of laminin fragments.
Adult human corneal epithelial basement membrane (EBM) and Descemet's membrane (DM) components exhibit heterogeneous distribution. The purpose of the study was to identify changes of these components during postnatal corneal development.
Thirty healthy adult corneas and 10 corneas from 12-day- to 3-year-old children were studied by immunofluorescence with antibodies against BM components.
Type IV collagen composition of infant corneal central EBM over Bowman's layer changed from α1-α2 to α3-α4 chains after 3 years of life; in the adult, α1-α2 chains were retained only in the limbal BM. Laminin α2 and β2 chains were present in the adult limbal BM where epithelial stem cells are located. By 3 years of age, β2 chain appeared in the limbal BM. In all corneas, limbal BM contained laminin γ3 chain. In the infant DM, type IV collagen α1-α6 chains, perlecan, nidogen-1, nidogen-2, and netrin-4 were found on both faces, but they remained only on the endothelial face of the adult DM. The stromal face of the infant but not the adult DM was positive for tenascin-C, fibrillin-1, SPARC, and laminin-332. Type VIII collagen shifted from the endothelial face of infant DM to its stromal face in the adult. Matrilin-4 largely disappeared after the age of 3 years.
The distribution of laminin γ3 chain, nidogen-2, netrin-4, matrilin-2, and matrilin-4 is described in the cornea for the first time. The observed differences between adult and infant corneal BMs may relate to changes in their mechanical strength, corneal cell adhesion and differentiation in the process of postnatal corneal maturation.
The laminin-5 component of the extracellular matrices of certain cultured cells such as the rat epithelial cell line 804G and the human breast epithelial cell MCF-10A is capable of nucleating assembly of cell– matrix adhesive devices called hemidesmosomes when other cells are plated upon them. These matrices also impede cell motility. In contrast, cells plated onto the laminin-5–rich matrices of pp126 epithelial cells fail to assemble hemidesmosomes and are motile. To understand these contradictory phenomena, we have compared the forms of heterotrimeric laminin-5 secreted by 804G and MCF-10A cells with those secreted by pp126 cells, using a panel of laminin-5 subunit-specific antibodies. The α3 subunit of laminin-5 secreted by pp126 cells migrates at 190 kD, whereas that secreted by 804G and MCF-10A cells migrates at 160 kD. The pp126 cell 190-kD α3 chain of laminin-5 can be specifically proteolyzed by plasmin to a 160-kD species at enzyme concentrations that do not apparently effect the laminin-5 β and γ chains. After plasmin treatment, pp126 cell laminin-5 not only impedes cell motility but also becomes competent to nucleate assembly of hemidesmosomes. The possibility that plasmin may play an important role in processing laminin-5 subunits is supported by immunofluorescence analyses that demonstrate colocalization of laminin-5 and plasminogen in the extracellular matrix of MCF-10A and pp126 cells. Whereas tissue-type plasminogen activator (tPA), which converts plasminogen to plasmin, codistributes with laminin-5 in MCF-10A matrix, tPA is not present in pp126 extracellular matrix. Treatment of pp126 laminin-5–rich extracellular matrix with exogenous tPA results in proteolysis of the laminin-5 α3 chain from 190 to 160 kD. In addition, plasminogen and tPA bind laminin-5 in vitro. In summary, we provide evidence that laminin-5 is a multifunctional protein that can act under certain circumstances as a motility and at other times as an adhesive factor. In cells in culture, this functional conversion appears dependent upon and is regulated by tPA and plasminogen.
The α4 laminin subunit is a component of endothelial cell
basement membranes. An antibody (2A3) against the α4 laminin G domain
stains focal contact-like structures in transformed and primary
microvascular endothelial cells (TrHBMECs and HMVECs, respectively),
provided the latter cells are activated with growth factors. The 2A3
antibody staining colocalizes with that generated by αv and β3
integrin antibodies and, consistent with this localization,
TrHBMECs and HMVECs adhere to the α4 laminin subunit G domain in an
αvβ3-integrin–dependent manner. The αvβ3
integrin/2A3 antibody positively stained focal contacts are
recognized by vinculin antibodies as well as by antibodies against
plectin. Unusually, vimentin intermediate filaments, in addition to
microfilament bundles, interact with many of the αvβ3
integrin-positive focal contacts. We have investigated the
function of α4-laminin and αvβ3-integrin, which are at
the core of these focal contacts, in cultured endothelial cells.
Antibodies against these proteins inhibit branching morphogenesis of
TrHBMECs and HMVECs in vitro, as well as their ability to repopulate in
vitro wounds. Thus, we have characterized an endothelial cell matrix
adhesion, which shows complex cytoskeletal interactions and whose
assembly is regulated by growth factors. Our data indicate that this
adhesion structure may play a role in angiogenesis.
Laminin-5 (LN5) is a matrix component of epithelial tissue basement membranes and plays an important role in the initiation and maintenance of epithelial cell anchorage to the underlying connective tissue. Here we show that two distinct LN5 function-inhibitory antibodies, both of which bind the globular domain of the α3 subunit, inhibit proliferation of epithelial cells. These same antibodies also induce a decrease in mitogen-activated protein kinase activity. Inhibition of proliferation by the function-perturbing LN5 antibodies is reversed upon removal of the antibodies and can be overcome by providing the antibody-treated cells with exogenous LN5 and rat tail collagen. Because epithelial cells use the integrin receptor α3β1 to interact with both LN5 and rat tail collagen, we next investigated the possibility that integrin α3β1 is involved in mediating the proliferative impact of LN5. Proliferation of human epithelial cells is significantly inhibited by a function-perturbing α3 integrin antibody. In addition, antibody activation of β1 integrin restores the proliferation of epithelial cells treated with LN5 function-perturbing antibodies. These data indicate that a complex comprising LN5 and α3β1 integrin is multifunctional and contributes not only to epithelial cell adhesion but also to the regulation of cell growth via a signaling pathway involving mitogen-activated protein kinase. We discuss our study in light of recent evidence that LN5 expression is up-regulated at the leading tips of tumors, where it may play a role in tumor cell proliferation.