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1.  Laminin-332 and α3β1 Integrin–Supported Migration of Bronchial Epithelial Cells Is Modulated by Fibronectin 
The repair of the bronchiolar epithelium damaged by cell-mediated, physical, or chemical insult requires epithelial cell migration over a provisional matrix composed of complexes of extracellular matrix molecules, including fibronectin and laminin. These matrix molecules support migration and enhance cell adhesion. When cells adhere too tightly to their matrix they fail to move; but if they adhere too little, they are unable to develop the traction force necessary for motility. Thus, we investigated the relative contributions of laminin and fibronectin to bronchiolar cell adhesion and migration using the immortalized bronchial lung epithelial cell line (BEP2D) and normal human bronchial epithelial (NHBE) cells, both of which assemble a laminin α3β3γ2 (LM332)/fibronectin-rich matrix. Intriguingly, BEP2D and NHBE cells migrate significantly faster on an LM332-rich matrix than on fibronectin. Moreover, addition of fibronectin to LM332 matrix suppresses motility of both cell types. Finally, fibronectin enhances the adhesion of both BEP2D and NHBE cells to LM332-coated surfaces. These results suggest that fibronectin fine tunes LM332-mediated migration by boosting bronchiolar cell adhesion to substrate. We suggest that, during epithelial wound healing of the injured airway, fibronectin plays an important adhesive role for laminin-driven epithelial cell motility by promoting a stable cellular interaction with the provisional matrix.
PMCID: PMC3931100  PMID: 23590307
motility; adhesion; extracellular matrix
2.  Focal Contact and Hemidesmosomal Proteins in Keratinocyte Migration and Wound Repair 
Advances in Wound Care  2014;3(3):247-263.
Significance: During wound healing of the skin, keratinocytes should move over while still adhering to their underlying matrix. Thus, mechanistic insights into the wound-healing process require an understanding of the forms and functions of keratinocyte matrix adhesions, specifically focal contacts and hemidesmosomes, and their components.
Recent Advances: Although the structure and composition of focal contacts and hemidesmosomes are relatively well defined, the functions of their components are only now being delineated using mouse genetic models and knockdown approaches in cell culture systems. Remarkably, both focal contact and hemidesmosomal proteins appear involved in determining the speed and directional migration of epidermal cells by modulating several signal transduction pathways.
Critical Issues: Although many publications are centered on focal contacts, their existence in tissues such as the skin is controversial. Nonetheless, focal contact proteins are central to mechanisms that regulate skin cell motility. Conversely, hemidesmosomes have been identified in intact skin but whether hemidesmosomal components play a positive regulatory function in keratinocyte motility remains debated in the field.
Future Directions: Defective wound healing is a developing problem in the aged, hospitalized and diabetic populations. Hence, deriving new insights into the molecular roles of matrix adhesion proteins in wound healing is a prerequisite to the development of novel therapeutics to enhance tissue repair and regeneration.
PMCID: PMC3955972  PMID: 24669360
3.  Flii Control: Balancing Migration and Adhesion 
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.
PMCID: PMC2863130  PMID: 19603052
4.  Alpha actinin-1 regulates cell-matrix adhesion organization in keratinocytes: consequences for skin cell motility 
The migration of keratinocytes in wound healing requires coordinated activities of the motility machinery of a cell, the cytoskeleton and matrix adhesions. In this study we assessed the role of alpha actinin-1 (ACTN1), one of the two alpha actinin isoforms expressed in keratinocytes, in skin cell migration via an shRNA-mediated knockdown approach. Keratinocytes deficient in ACTN1 exhibit changes in their actin cytoskeleton organization, a loss in front-rear polarity and impaired lamellipodial dynamics. They also display aberrant directed motility and move slower than their wild-type counterparts. Moreover, they have abnormally arranged matrix adhesion sites. Specifically, the focal adhesions in ACTN1 knockdown keratinocytes are not organized as distinct entities. Rather, focal adhesion proteins are arranged in a circle subjacent to cortical fibers of actin. In the same cells, hemidesmosome proteins arrange in cat paw patterns, more typical of confluent, stationary cells and β4 integrin dynamics are reduced in knockdown cells compared with control keratinocytes. In summary, our data suggest a mechanism by which ACTN1 determines the motility of keratinocytes by regulating the organization of the actin cytoskeleton, focal adhesion and hemidesmosome proteins complexes, thereby modulating cell speed, lamellipodial dynamics and directed migration.
PMCID: PMC4366307  PMID: 25431851
motility; lamellipodia; focal adhesion; hemidesmosome; integrin
5.  BPAG1e Maintains Keratinocyte Polarity through β4 Integrin–mediated Modulation of Rac 1 and Cofilin Activities 
Molecular Biology of the Cell  2009;20(12):2954-2962.
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.
PMCID: PMC2695802  PMID: 19403692
6.  Hemidesmosomes and focal contact proteins: Functions and cross-talk in keratinocytes, bullous diseases and wound healing 
The outer most layer of the skin, the epidermis, is attached to the dermis via a sheet of extracellular matrix proteins termed the basement membrane zone (BMZ). In the intact skin, adhesion of the keratinocytes in the basal layer of the epidermis to the BMZ is facilitated primarily by hemidesmosomes which associate with the keratin cytoskeleton. Cultured keratinocytes do not assemble bona fide hemidesmosomes although hemidesmosome protein clusters (stable anchoring contacts) are found along the substrate-attached surface of the cells and towards the leading edge of keratinocytes repopulating scratch wounds. Actin cytoskeleton-associated matrix adhesion devices termed focal contacts are not thought to play an important role in the adhesion of keratinocytes to the BMZ in intact skin but are prominent in cultured keratinocytes where they are believed to regulate cell migration. We review the molecular components, functions, dynamics and cross-talk of hemidesmosomes and focal contacts in keratinocytes. In addition, we briefly describe what is known about their role in autoimmune and genetic blistering diseases of the skin. We also discuss recent publications which indicate, contrary to expectation, that certain focal contact proteins retard keratinocyte migration while hemidesmosomal proteins regulate directed keratinocyte motility during wound healing.
PMCID: PMC4492441  PMID: 21376539
Matrix adhesion; Focal adhesion; Focal complex; Bullous pemphigoid; Epidermolysis bullosa; Wound repair
7.  A New Component of the Fraser Complex 
In embryos, the Fraser Complex (FC) mediates epithelial-connective tissue interactions. Loss of expression of FC components leads to Fraser Syndrome (FS) in which cohesion of epithelial tissues and stroma is perturbed. Using zebrafish, Richardson et al (in this issue) identified the protein AMACO in the FC. We discuss the utility of zebrafish in determining FC functions and identifying FS targets.
PMCID: PMC4007285  PMID: 24732331
8.  A Role for Anti-BP180 Autoantibodies in Chronic Rhinosinusitis 
The Laryngoscope  2013;123(9):2104-2111.
CRS is accompanied by evidence of a vigorous adaptive immune response, and emerging studies demonstrate that some nasal polyps manifest a polyclonal autoantibody response. We previously found that antibodies against BP180, a component of the hemidesmosome complex and the dominant epitope in autoimmune bullous pemphigoid, were found at elevated levels in nasal polyp tissue. Given the critical role of hemidesmosomes in maintaining epithelial integrity, we sought to investigate the distribution of BP180 in nasal tissue and evaluate for evidence of systemic autoimmunity against this antigen in CRS.
Study Design
Case-control experimental study
The expression and distribution of BP180 in cultured nasal epithelial cells and normal nasal tissue were confirmed using real-time PCR, Western immunoblotting, immunofluorescence and immunohistochemistry. Sera were collected from three groups: control, CRSsNP, and CRSwNP. A commercially available ELISA was utilized to compare anti-BP180 autoantibody levels in sera.
BP180 is expressed in nasal epithelium, but is not confined to the basement membrane as it is in human skin. In cultured nasal epithelial cells, confocal immunofluoresence showed a punctate distribution of BP180 along the basal surface, consistent with its distribution in epithelial keratinocytes. There are significantly higher levels of circulating nonpathologic anti-BP180 autoantibodies in CRS patients compared with normal controls (p<0.05).
BP180 is more widely expressed in nasal epithelium versus skin, although it appears to play a similar role in formation of hemidesmosomes along the basement membrane. Further investigations are ongoing to characterize the pathogenicity of the anti-epithelial antibody response in CRS.
PMCID: PMC3813294  PMID: 24167818
Chronic rhinosinusitis; sinusitis; nasal polyps; autoimmunity; autoantibodies; biomarker; bullous pemphigoid
9.  Fibronectin expression determines skin cell motile behavior 
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.
PMCID: PMC3252482  PMID: 21956124
10.  Transdominant Regulation of Integrin Function: Mechanisms of Crosstalk 
Cellular signalling  2009;22(4):578.
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.
PMCID: PMC2814898  PMID: 19874888
Matrix adhesion; receptors; cytoskeleton; signaling; adaptor proteins; phosphorylation
11.  Spatial and Temporal Control of Laminin-332 (5) and -511 (10) Expression During Induction of Anagen Hair Growth 
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.
PMCID: PMC3046633  PMID: 16957169
laminin; hemidesmosome; integrin; protease; hair cycle
12.  Adhesion and Migration, the Diverse Functions of the Laminin α3 Subunit 
Dermatologic clinics  2010;28(1):79.
PMCID: PMC2814596  PMID: 19945619
13.  Laminin-332-Integrin Interaction: A Target For Cancer Therapy? 
Current medicinal chemistry  2008;15(20):1968-1975.
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.
PMCID: PMC2992754  PMID: 18691052
Integrin; laminin; cancer; basement membrane; proteolysis; cell signaling; cell adhesion; gene expression
14.  14-3-3 Sigma Isoform Interacts with the Cytoplasmic Domain of the Transmembrane BP180 in Keratinocytes 
Journal of cellular physiology  2007;212(3):675-681.
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.
PMCID: PMC2991636  PMID: 17443672
15.  Wound Healing Is Defective in Mice Lacking Tetraspanin CD151 
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.
PMCID: PMC2976039  PMID: 16410781
16.  14-3-3ζ/τ heterodimers regulate Slingshot activity in migrating keratinocytes 
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.
PMCID: PMC2727353  PMID: 19371722
Slingshot; 14-3-3; migration; keratinocytes
17.  Dynamic Relationship of Focal Contacts and Hemidesmosome Protein Complexes in Live Cells 
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.
PMCID: PMC2882194  PMID: 20107487
18.  Laminin-511, inducer of hair growth, is down-regulated and its suppressor in hair growth, laminin-332 up-regulated in chemotherapy-induced alopecia 
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.
PMCID: PMC2864493  PMID: 20211547
A549 cells; Chemotherapy; Extracellular matrix; Hemidesmosome; Integrin; Outer root sheath cells
19.  Recruitment of vimentin to the cell surface by β3 integrin and plectin mediates adhesion strength 
Journal of cell science  2009;122(Pt 9):1390-1400.
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.
PMCID: PMC2721003  PMID: 19366731
Intermediate filament; Integrin; Adhesion
20.  Integrin β4 Regulates Migratory Behavior of Keratinocytes by Determining Laminin-332 Organization*s 
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.
PMCID: PMC2820731  PMID: 16973601
21.  Laminin-6 assembles into multimolecular fibrillar complexes with perlecan and participates in mechanical-signal transduction via a dystroglycan-dependent, integrin-independent mechanism 
Journal of cell science  2005;118(Pt 12):2557.
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.
PMCID: PMC2820238  PMID: 15928048
Matrix adhesion; Matrix receptors; Stretching
22.  Caspase Proteolysis of the Integrin β4 Subunit Disrupts Hemidesmosome Assembly, Promotes Apoptosis, and Inhibits Cell Migration* 
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.
PMCID: PMC2819670  PMID: 17178732
23.  β4 integrin-dependent formation of polarized three-dimensional architecture confers resistance to apoptosis in normal and malignant mammary epithelium 
Cancer cell  2002;2(3):205-216.
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.
PMCID: PMC2788997  PMID: 12242153
24.  Stretch-Induced Activation of AMP Kinase in the Lung Requires Dystroglycan 
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.
PMCID: PMC2586043  PMID: 18556591
stretch; lung injury; mechanical ventilation; acute respiratory distress syndrome
25.  The Slingshot Family of Phosphatases Mediates Rac1 Regulation of Cofilin Phosphorylation, Laminin-332 Organization, and Motility Behavior of Keratinocytes*s 
The Journal of biological chemistry  2007;282(44):32520-32528.
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.
PMCID: PMC2754063  PMID: 17848544

Results 1-25 (32)