Fas palmitoylation by the palmitoyl acyltransferase DHHC7 regulates Fas stability.

The death receptor Fas undergoes a variety of post-translational modifications including S-palmitoylation. This protein acylation has been reported essential for an optimal cell death signaling by allowing both a proper Fas localization in cholesterol and sphingolipid-enriched membrane nanodomains, as well as Fas high-molecular weight complexes. In human, S-palmitoylation is controlled by 23 members of the DHHC family through their palmitoyl acyltransferase activity. In order to better understand the role of this post-translational modification in the regulation of the Fas-mediated apoptosis pathway, we performed a screen that allowed the identification of DHHC7 as a Fas-palmitoylating enzyme. Indeed, modifying DHHC7 expression by specific silencing or overexpression, respectively, reduces or enhances Fas palmitoylation and DHHC7 co-immunoprecipitates with Fas. At a functional level, DHHC7-mediated palmitoylation of Fas allows a proper Fas expression level by preventing its degradation through the lysosomes. Indeed, the decrease of Fas expression obtained upon loss of Fas palmitoylation can be restored by inhibiting the lysosomal degradation pathway. We describe the modification of Fas by palmitoylation as a novel mechanism for the regulation of Fas expression through its ability to circumvent its degradation by lysosomal proteolysis.

EGF-R signaling through Fyn kinase disrupts the function of integrin alpha6beta4 at hemidesmosomes: role in epithelial cell migration and carcinoma invasion.

We have examined the mechanism and functional significance of hemidesmosome disassembly during normal epithelial cell migration and squamous carcinoma invasion. Our findings indicate that a fraction of EGF receptor (EGF-R) combines with the hemidesmosomal integrin alpha6beta4 in both normal and neoplastic keratinocytes. Activation of the EGF-R causes tyrosine phosphorylation of the beta4 cytoplasmic domain and disruption of hemidesmosomes. The Src family kinase inhibitors PP1 and PP2 prevent tyrosine phosphorylation of beta4 and disassembly of hemidesmosomes without interfering with the activation of EGF-R. Coimmunoprecipitation experiments indicate that Fyn and, to a lesser extent, Yes combine with alpha6beta4. By contrast, Src and Lck do not associate with alpha6beta4 to a significant extent. A dominant negative form of Fyn, but not Src, prevents tyrosine phosphorylation of beta4 and disassembly of hemidesmosomes. These observations suggest that the EGF-R causes disassembly of hemidesmosomes by activating Fyn, which in turn phosphorylates the beta4 cytoplasmic domain. Neoplastic cells expressing dominant negative Fyn display increased hemidesmosomes and migrate poorly in vitro in response to EGF. Furthermore, dominant negative Fyn decreases the ability of squamous carcinoma cells to invade through Matrigel in vitro and to form lung metastases following intravenous injection in nude mice. These results suggest that disruption of hemidesmosomes mediated by Fyn is a prerequisite for normal keratinocyte migration and squamous carcinoma invasion.

The short arm of the laminin gamma2 chain plays a pivotal role in the incorporation of laminin 5 into the extracellular matrix and in cell adhesion.

Laminin 5 is a basement membrane component that actively promotes adhesion and migration of epithelial cells. Laminin 5 undergoes extracellular proteolysis of the gamma2 chain that removes the NH(2)-terminal short arm of the polypeptide and reduces the size of laminin 5 from 440 to 400 kD. The functional consequence of this event remains obscure, although lines of evidence indicate that cleavage of the gamma2 chain potently stimulated scattering and migration of keratinocytes and cancer cells. To define the biological role of the gamma2 chain short arm, we expressed mutated gamma2 cDNAs into immortalized gamma2-null keratinocytes. By immunofluorescence and immunohistochemical studies, cell detachment, and adhesion assays, we found that the gamma2 short arm drives deposition of laminin 5 into the extracellular matrix (ECM) and sustains cell adhesion. Our results demonstrate that the unprocessed 440-kD form of laminin 5 is a biologically active adhesion ligand, and that the gamma2 globular domain IV is involved in intermolecular interactions that mediate integration of laminin 5 in the ECM and cell attachment.

Tyrosine phosphorylation of the beta 4 integrin cytoplasmic domain mediates Shc signaling to extracellular signal-regulated kinase and antagonizes formation of hemidesmosomes.

Ligation of the alpha(6)beta(4) integrin induces tyrosine phosphorylation of the beta(4) cytoplasmic domain, followed by recruitment of the adaptor protein Shc and activation of mitogen-activated protein kinase cascades. We have used Far Western analysis and phosphopeptide competition assays to map the sites in the cytoplasmic domain of beta(4) that are required for interaction with Shc. Our results indicate that, upon phosphorylation, Tyr(1440), or secondarily Tyr(1422), interacts with the SH2 domain of Shc, whereas Tyr(1526), or secondarily Tyr(1642), interacts with its phosphotyrosine binding (PTB) domain. An inactivating mutation in the PTB domain of Shc, but not one in its SH2 domain, suppresses the activation of Shc by alpha(6)beta(4). In addition, mutation of beta(4) Tyr(1526), which binds to the PTB domain of Shc, but not of Tyr(1422) and Tyr(1440), which interact with its SH2 domain, abolishes the activation of ERK by alpha(6)beta(4). Phenylalanine substitution of the beta(4) tyrosines able to interact with the SH2 or PTB domain of Shc does not affect incorporation of alpha(6)beta(4) in the hemidesmosomes of 804G cells. Exposure to the tyrosine phosphatase inhibitor orthovanadate increases tyrosine phosphorylation of beta4 and disrupts the hemidesmosomes of 804G cells expressing recombinant wild type beta(4). This treatment, however, exerts a decreasing degree of inhibition on the hemidesmosomes of cells expressing versions of beta(4) containing phenylalanine substitutions at Tyr(1422) and Tyr(1440), at Tyr(1526) and Tyr(1642), or at all four tyrosine phosphorylation sites. These results suggest that beta(4) Tyr(1526) interacts in a phosphorylation-dependent manner with the PTB domain of Shc. This event is required for subsequent tyrosine phosphorylation of Shc and signaling to ERK but not formation of hemidesmosomes.

Role of the bullous pemphigoid antigen 180 (BP180) in the assembly of hemidesmosomes and cell adhesion--reexpression of BP180 in generalized atrophic benign epidermolysis bullosa keratinocytes.

Bullous pemphigoid antigen 180 (BP180) is a transmembrane component of hemidesmosomes (HD), cell-substrate attachment complexes in stratified and complex epithelia. To determine the role of BP180 in the assembly of HD and cell adhesion, using SV40 virions we have immortalized BP180-deficient keratinocytes derived from a patient with the inherited skin blistering disorder generalized atrophic benign epidermolysis bullosa (GABEB). The GABEB keratinocytes form HD-like structures, which contain alpha 6 beta 4 integrin and HD1/plectin, but not the bullous pemphigoid antigen 230 (BP230). The expression of integrin subunits by GABEB keratinocytes was comparable to that of an immortalized normal human keratinocyte cell line (NHK), except for alpha 6 and beta 4, which were less strongly expressed in GABEB cells. In short-term adhesion assays, both GABEB keratinocytes and NHK bound strongly and to a similar extent to laminin-1, laminin-5, fibronectin, and type IV and V collagens, which suggests that BP180 is not involved in promoting the initial adhesion to these ligands. Transfection of GABEB keratinocytes with cDNAs for wild-type or a mutant of BP180 lacking the collagenous extracellular domain resulted in the expression of recombinant BP180 proteins that were correctly polarized at the basal cell surface together with alpha 6 beta 4. In addition, restored synthesis of BP180 affected the subcellular localization of BP230, which was no longer diffusely distributed in the cytoplasm, but was found in HD-like structures. In contrast, a BP180 mutant with a 36-amino-acid deletion from the amino terminus of the cytoplasmic domain failed to localize to HD-like structures. These results demonstrate that a region within the cytoplasmic domain of BP180 is essential for its localization into HD and that BP180 may play a critical role in coordinating the subcellular distribution of BP230.

Compound heterozygosity for an out-of-frame deletion and a splice site mutation in the LAMB3 gene causes nonlethal junctional epidermolysis bullosa.

Laminin-5 is the major adhesion ligand of epithelial cells. Mutations in the genes encoding laminin-5 cause junctional epidermolysis bullosa (JEB), a clinically and genetically heterogeneous group of recessively inherited blistering disease of skin and mucous membranes. In this report, we describe a patient with a non-lethal variant of JEB who is a compound heterozygous for mutations affecting the LAMB3 gene. The paternally inherited mutation is a deletion of a single base (T) leading to a frameshift and premature termination codon. It results in mRNA decay. The maternally inherited mutation is a G-->A transition at the last base of exon 7 (628G-->A) which converts a codon for glutamic acid in a codon for lysine (E210K). The mutation 628G-->A alters the correct splicing of LAMB3 pre-mRNA giving rise to two aberrant mRNA, in addition to the RNA transcript carrying the G-->A substitution. This result is compatible with the reduced expression of mutated laminin 5 molecules with altered biological activity, and the mild JEB phenotype observed in the patient.

Corrective gene transfer of keratinocytes from patients with junctional epidermolysis bullosa restores assembly of hemidesmosomes in reconstructed epithelia.

Herlitz junctional epidermolysis bullosa (H-JEB) provides a promising model for somatic gene therapy of heritable mechano-bullous disorders. This genodermatosis is caused by the lack of laminin-5 that results in absence of hemidesmosomes (HD) and defective adhesion of squamous epithelia. To establish whether re-expression of laminin-5 can restore assembly of the dermal-epidermal attachment structures lacking in the H-JEB skin, we corrected the genetic mutation hindering expression of the beta 3 chain of laminin-5 in human H-JEB keratinocytes by transfer of a laminin beta 3 transgene. The transduced keratinocytes synthesized a recombinant beta 3 polypeptide that assembled with the endogenous laminin alpha 3 and gamma 2 chains into a biologically active laminin-5 that was secreted, processed and deposited into the extracellular matrix. Re-expression of laminin-5 induced cell spreading, nucleation of hemidesmosomal-like structures and enhanced adhesion to the culture substrate. Organotypic cultures performed with the transduced keratinocytes, reconstituted epidermis closely adhering to the mesenchyme and presenting mature hemidesmosomes, bridging the cytoplasmic intermediate filaments of the basal cells to the anchoring filaments of the basement membrane. Our results provide the first evidence of phenotypic reversion of JEB keratinocytes by somatic gene therapy and demonstrate that genetic treatment of the mild forms of skin blistering diseases and other inherited extracellular matrix pathologies is a realistic goal.

Hemidesmosome assembly assessed by expression of a wild-type integrin beta 4 cDNA in junctional epidermolysis bullosa keratinocytes.

The cytoplasmic domain of integrin beta 4, which contains four type III fibronectin-like motifs, seems to be involved in the regulation of the assembly of hemidesmosomes (HD) and, therefore, in cell adhesion. An in-frame deletion of 17 amino acids in the second fibronectin type III repeat of integrin beta 4 (delta 17-beta 4) has been associated with junctional epidermolysis bullosa with pyloric atresia (PA-JEB), a genetic disease characterized by altered HD and disadhesion of the epidermis. To determine the effect of deletion delta 17-beta 4 on HD assembly, we have examined the expression and localization of the HD components in the skin and cultured keratinocytes of a patient with PA-JEB, which express the mutated integrin beta 4. Our results show that the mutated beta 4 subunit associates with integrin alpha 6, but the resulting heterodimer does not induce nucleation of the bullous pemphigoid antigens BP180 and BP230, and that of the inner plaque component plectin/HD1, into hemidesmosomal structures. The integrity of the cytoplasmic tail of integrin beta 4 seems to be essential to the targeting and stabilization of plectin/HD1 and BP180 in HD, because transfection of a recombinant wild-type B4 cDNA in the delta 17-beta 4 PA-JEB keratinocytes restores the synthesis of a functional alpha 6/beta 4 heterodimer, which promotes the polarization of plectin/HD1 and BP180, to the basal aspect of the cells. Because in the transfected keratinocytes the distribution of BP230 remains diffuse in the cytoplasm, we suggest that the interaction between plectin/HD1 and integrin alpha 6 beta 4, followed by the association with BP180, constitutes the first step in the nucleation of the HD.

A homozygous mutation in the integrin alpha6 gene in junctional epidermolysis bullosa with pyloric atresia.

The alpha6 integrin subunit participates in the formation of both alpha6beta1 and alpha6beta4 laminin receptors, which have been reported to play an important role in cell adhesion and migration and in morphogenesis. In squamous epithelia, the alpha6beta4 heterodimer is the crucial component for the assembly and stability of hemidesmosomes. These anchoring structures are ultrastructurally abnormal in patients affected with junctional epidermolysis bullosa with pyloric atresia (PA-JEB), a recessively inherited blistering disease of skin and mucosae characterized by an altered immunoreactivity with antibodies specific to integrin alpha6beta4. In this report, we describe the first mutation in the alpha6 integrin gene in a PA-JEB patient presenting with generalized skin blistering, aplasia cutis, and defective expression of integrin alpha6beta4. The mutation (791delC) is a homozygous deletion of a single base (C) leading to a frameshift and a premature termination codon that results in a complete absence of alpha6 polypeptide. We also describe the DNA-based prenatal exclusion of the disease in this family at risk for recurrence of PA-JEB. Our results demonstrate that, despite the widespread distribution of the alpha6 integrin subunit, lack of expression of the alpha6 integrin chain is compatible with fetal development, and results in a phenotype indistinguishable from that caused by mutations in the beta4 chain, which is expressed in a more limited number of tissues.

Functional Re-expression of laminin-5 in laminin-gamma2-deficient human keratinocytes modifies cell morphology, motility, and adhesion.

Herlitz junctional epidermolysis bullosa (H-JEB) is characterized by a reduced adherence of keratinocytes consequent to deficient expression of the extracellular adhesive ligand laminin-5. To complement the genetic defect causing H-JEB, we transferred an eukaryotic cassette expressing the cDNA for the gamma2 chain of laminin-5 into H-JEB keratinocytes in which the expression of the polypeptide is hampered by a homozygous mutation generating a premature termination codon. Transfection using adenovirus-polylysin-transferrin-DNA complexes resulted in a transient synthesis of the recombinant laminin gamma2 chain that associated with the endogenous alpha3 and beta3 chains to form laminin-5 molecules readily deposited on the tissue culture substrate. Furthermore, retroviral-mediated transduction of the gamma2 cDNA yielded persistent expression and polarized secretion of laminin-5. The protein incorporated into the basement membrane produced by the revertant cells inoculated subcutaneously in nude mice. In these transfectants, re-expression of laminin-5 induced changes in cell morphology and reorganization of focal adhesions that assumed the shape and distribution of the counterparts detected in normal keratinocytes. These observations correlated with an enhanced cell-substrate adhesion and a reduced motility of the transfected cells. Our results demonstrate that a restored expression of laminin-5 induces a phenotypic reversion of genetically altered H-JEB keratinocytes and open new perspectives to the analysis of the mechanisms regulating adhesion of epithelial cells.

Establishment and characterization of cell line LSV5 that retains the altered adhesive properties of human junctional epidermolysis bullosa keratinocytes.

Herlitz junctional epidermolysis bullosa (H-JEB) is characterized by hampered expression of the adhesion ligand laminin-5. Thus far, analysis of the processes underlying the epithelial-mesenchymal dysadhesion marking this disease has been limited by the reduced growth and adhesive capabilities of the epithelial cells derived from H-JEB patients. To overcome these difficulties, we used SV40 virus to immortalize H-JEB keratinocytes with a homozygous nonsense mutation in the gene that encodes the gamma2 chain of laminin-5. Cell lines (LSV) derived from infected keratinocytes maintain a stable karyotype, grow independent of 3T3 feeder layers and are not tumorigenic. Further analysis of clone LSV5 showed an increased secretion of laminin-6 and fibronectin compared to normal keratinocytes. Similar to parental H-JEB keratinocytes, these cells regenerate stratified epidermis in vitro and, in in vivo models, they synthesize a basement membrane lacking laminin-5. LSV cells show hypermotility and reduced adhesive properties resulting from an incomplete association with the underlying culture substrate. These results demonstrate that LSV5 cells retain the pathologic phenotype of H-JEB keratinocytes and can serve as a model system to study the adhesion processes mediated by laminin-5.