Gene correction of integrin beta4-dependent pyloric atresia-junctional epidermolysis bullosa keratinocytes establishes a role for beta4 tyrosines 1422 and 1440 in hemidesmosome assembly.

Elena Dellambra,Anna Laura Salvati,Michele De Luca,Giovanna Zambruno,Maria Luisa Madeddu,Filippo G Giancotti,Sergio Bondanza,Emanuela Siviero,Silvia Prislei,Sandra Cicuzza,Angela Orecchia,Osvaldo Golisano

doi:10.1074/jbc.m103139200

Abstract

The cytoplasmic domain of beta4 integrin contains two pairs of fibronectin-like repeats separated by a connecting segment. The connecting segment harbors a putative tyrosine activation motif in which tyrosines 1422 and 1440 are phosphorylated in response to alpha6beta4 binding to laminin-5. Primary beta4-null keratinocytes, obtained from a newborn suffering from lethal junctional epidermolysis bullosa, were stably transduced with retroviruses carrying a full-length beta4 cDNA or a beta4 cDNA with phenylalanine substitutions at Tyr-1422 and Tyr-1440. Hemidesmosome assembly was evaluated on organotypic skin cultures. beta4-corrected keratinocytes were indistinguishable from normal cells in terms of alpha6beta4 expression, the localization of hemidesmosome components, and hemidesmosome structure and density, suggesting full genetic and functional correction of beta4-null keratinocytes. In cultures generated from beta4(Y1422F/Y1440F) keratinocytes, beta4 mutants as well as alpha6 integrin, HD1/plectin, and BP180 were not concentrated at the dermal-epidermal junction. Furthermore, the number of hemidesmosomes was strikingly reduced as compared with beta4-corrected keratinocytes. The rare hemidesmosomes detected in beta4(Y1422F/Y1440F) cells were devoid of sub-basal dense plates and of inner cytoplasmic plaques with keratin filament insertion. Collectively, our data demonstrate that the beta4 tyrosine activation motif is not required for the localization of alpha6beta4 at the keratinocyte plasma membrane but is essential for optimal assembly of bona fide hemidesmosomes.

Highlights

The cytoplasmic domain of ␤4 integrin contains two pairs of fibronectin-like repeats separated by a connecting segment
In addition to resolving the prior controversy, these results reveal a functional synergy between amino acid stretches located relatively far apart in the linear sequence of the ␤4 cytoplasmic domain and highlight the necessity to further examine the potential role of the ␤4 tyrosine activation motif (TAM) in HD assembly
Keratinocytes were cultivated from a 1-cm2 skin biopsy taken from a newborn patient presenting with the clinical hallmarks of pyloric atresia (PA)-junctional epidermolysis bullosa (JEB)

Summary

In cultures generated from

Plectin, and BP180 were not concentrated at the dermalepidermal junction. the number of hemidesmosomes was strikingly reduced as compared with ␤4-corrected keratinocytes. The potential role of specific portions of the ␤4 cytoplasmic domain, and in particular of the TAM, in interaction with other HD components and in HD assembly is based solely on the results obtained using immortalized cell lines cultured on plastic Under these conditions, both keratinocytes and 804G cells do not form HDs. Instead, HD components (such as ␣6␤4, BP180, and HD1/plectin) are organized in typical patches in which spots correspond to microfilament-free areas (“leopard skin” pattern, as described in Ref. 24), often referred to as HD-like adhesions [22, 25]. The availability of human ␤4-deficient primary keratinocytes (see “Results”), the possibility of stably transducing primary keratinocytes with high efficiency [34], and the possibility of subcultivating stably transduced cells in conditions in which HDs are formed [30] provide a unique opportunity to clarify the above uncertainties and to investigate the role of ␤4 and of its potential TAM in the formation of mature HDs

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION

Cell strain