Kevin J. Hamill

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.

Laminin deposition in the extracellular matrix: a complex picture emerges.

Laminins are structural components of basement membranes. In addition, they are key extracellular-matrix regulators of cell adhesion, migration, differentiation and proliferation. This Commentary focuses on a relatively understudied aspect of laminin biology: how is laminin deposited into the extracellular matrix? This topic has fascinated researchers for some time, particularly considering the diversity of patterns of laminin that can be visualized in the matrix of cultured cells. We discuss current ideas of how laminin matrices are assembled, the role of matrix receptors in this process and how laminin-associated proteins modulate matrix deposition. We speculate on the role of signaling pathways that are involved in laminin-matrix deposition and on how laminin patterns might play an important role in specifying cell behaviors, especially directed migration. We conclude with a description of new developments in the way that laminin deposition is being studied, including the use of tagged laminin subunits that should allow the visualization of laminin-matrix deposition and assembly by living cells.

BPAG1e Maintains Keratinocyte Polarity through β4 Integrin–mediated Modulation of Rac 1 and Cofilin Activities

alpha6beta4 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 beta4 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 beta4 integrin expressed at the cell surface. However, the amount of Rac1 associating with beta4 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.

A role for anti-BP180 autoantibodies in chronic rhinosinusitis

Chronic Rhinosinusitis (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.

Adhesion and Migration, the Diverse Functions of the Laminin α3 Subunit

The laminins are a secreted family of heterotrimeric molecules essential for basement membrane formation, structure, and function. It is now well established that the alpha3 subunit of laminins-332, -321, and -311 plays an important role in mediating epidermal-dermal integrity and is essential for the skin to withstand mechanical stresses. These laminins also regulate cell migration and mechanosignal transduction. This article provides an overview of the gene, transcripts, and protein structures of laminin alpha3. Also discussed are the proposed functions for the alpha3 subunit-containing laminins.

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.

Type XVII collagen regulates lamellipod stability, cell motility, and signaling to Rac1 by targeting bullous pemphigoid antigen 1e to alpha6beta4 integrin.

Rac1 activity, polarity, lamellipodial dynamics, and directed motility are defective in keratinocytes exhibiting deficiency in β4 integrin or knockdown of the plakin protein Bullous Pemphigoid Antigen 1e (BPAG1e). The activity of Rac, formation of stable lamellipodia, and directed migration are restored in β4 integrin-deficient cells by inducing expression of a truncated form of β4 integrin, which lacks binding sites for BPAG1e and plectin. In these same cells, BPAG1e, the truncated β4 integrin, and type XVII collagen (Col XVII), a transmembrane BPAG1e-binding protein, but not plectin, colocalize along the substratum-attached surface. This finding suggested to us that Col XVII mediates the association of BPAG1e and α6β4 integrin containing the truncated β4 subunit and supports directed migration. To test these possibilities, we knocked down Col XVII expression in keratinocytes expressing both full-length and truncated β4 integrin proteins. Col XVII-knockdown keratinocytes exhibit a loss in BPAG1e-α6β4 integrin interaction, a reduction in lamellipodial stability, an impairment in directional motility, and a decrease in Rac1 activity. These defects are rescued by a mutant Col XVII protein truncated at its carboxyl terminus. In summary, our results suggest that in motile cells Col XVII recruits BPAG1e to α6β4 integrin and is necessary for activation of signaling pathways, motile behavior, and lamellipodial stability.

Actinin-4 in keratinocytes regulates motility via an effect on lamellipodia stability and matrix adhesions

During wound repair, epidermal cells at the edge of an injury establish front‐rear polarity through orchestrated changes in their cytoskeleton and adhesion structures. The polarity and directed migration of such cells is determined by the assembly, extension, and stabilization of a lamellipodium. Actinin‐4 associates with lamellipodia and has been implicated in regulating lamellipodial structure, function and assembly. To study the functions of actinin‐4 in human keratinocytes, we used shRNA to generate knockdown cells and compared their motility behavior and matrix adhesion assembly to scrambled shRNA treated control keratinocytes. Actinin‐4 knockdown keratinocytes lack polarity, assemble multiple lamellipodia with a 2× increased area over controls, display reduced activity of the actin remodeling protein cofilin, and fail to migrate in a directional manner. This motility defect is rescued by plating knockdown cells on preformed laminin‐332 matrix. In actinin‐4‐knockdown keratinocytes, focal contact area is increased by 25%, and hemidesmosome proteins are mislocalized. Specifically, α6β4 integrin localizes to large lamellipodial extensions, displays reduced dynamics, and fails to recruit its bullous pemphigoid antigen binding partners. Together, our data indicate a role for actinin‐4 in regulating the steering mechanism of keratinocytes via profound effects on their matrix adhesion sites.—Hamill, K. J., Hopkinson, S. B., Skalli, O., Jones, J. C. R. Actinin‐4 in keratinocytes regulates motility via an effect on lamellipodia stability and matrix adhesions. FASEB J. 27, 546–556 (2013). www.fasebj.org

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.

Lung-Specific Loss of α3 Laminin Worsens Bleomycin-Induced Pulmonary Fibrosis

Laminins are heterotrimeric proteins that are secreted by the alveolar epithelium into the basement membrane, and their expression is altered in extracellular matrices from patients with pulmonary fibrosis. In a small number of patients with pulmonary fibrosis, we found that the normal basement membrane distribution of the α3 laminin subunit was lost in fibrotic regions of the lung. To determine if these changes play a causal role in the development of fibrosis, we generated mice lacking the α3 laminin subunit specifically in the lung epithelium by crossing mice expressing Cre recombinase driven by the surfactant protein C promoter (SPC-Cre) with mice expressing floxed alleles encoding the α3 laminin gene (Lama3(fl/fl)). These mice exhibited no developmental abnormalities in the lungs up to 6 months of age, but, compared with control mice, had worsened mortality, increased inflammation, and increased fibrosis after the intratracheal administration of bleomycin. Similarly, the severity of fibrosis induced by an adenovirus encoding an active form of transforming growth factor-β was worse in mice deficient in α3 laminin in the lung. Taken together, our results suggest that the loss of α3 laminin in the lung epithelium does not affect lung development, but plays a causal role in the development of fibrosis in response to bleomycin or adenovirally delivered transforming growth factor-β. Thus, we speculate that the loss of the normal basement membrane organization of α3 laminin that we observe in fibrotic regions from the lungs of patients with pulmonary fibrosis contributes to their disease progression.