Isolation and characterization of a cDNA clone coding for human epidermal keratin K5. Sequence of the carboxyterminal half of this keratin.

A cDNA clone encoding human epidermal keratin No. 5 (K5) has been isolated from a lambda gt10 cDNA library on the basis of crosshybridization with cDNAs coding for other basic keratins and differential hybridization with total cDNA probes. This clone contains a 1100-bp insert able to inhibit specifically translation of K5 in a hybrid-arrested translation test. Northern blots show that this insert hybridizes with a 2.4-kb message present in epidermal mRNAs. The 1100-bp sequence reported here corresponds to the 3' half of the K5 message. Comparison of the deduced aminoacid sequence shows that the protein exhibits characteristic features of a basic keratin. The 242 aminoacid sequence reported here extends from the carboxyterminal end up to the last helical portion of the central rod domain.

Abnormal sequence of expression of differentiation markers in psoriatic epidermis: inversion of two steps in the differentiation program?

This immunohistologic study was undertaken to compare epidermal differentiation in normal and psoriatic skin. Although basal cells retain a normal phenotype in this disease, suprabasal layers exhibit abnormal sets of differentiation markers. The 67-kD keratin and Bd5 antigen, which are found in normal epidermis immediately above the basal layer, appear several layers higher in involved psoriatic epidermis. On the contrary, KF2 antigen, which is found in the upper spinous layers of normal epidermis, appears more precociously in psoriatic epidermis. Paradoxically, in this disease characterized by the absence of a granular layer, some markers specific for this layer in normal skin, such as involucrin and transglutaminase, appear in lower skin cell layers, while other granular markers, such as filaggrin, are either absent or found in the parakeratotic scales. These results point out the existence in psoriasis of a suprabasal cell population characterized by a set of markers that are never coexpressed in normal epidermis. The existence of this abnormal population of cells can be explained as the result of the inversion of two steps in the differentiation program. Thus, instead of an inability to express a given differentiation marker, psoriasis seems to be characterized by an abnormal sequence of expression of these markers.

Spreading of psoriatic plaques: alteration of epidermal differentiation precedes capillary leakiness and anomalies in vascular morphology.

To approach the temporal relationship between alterations in keratinization and capillary leakiness in psoriasis, we studied the topography of these anomalies in spreading psoriatic lesions. Histological and immunohistochemical studies were performed on skin biopsies obtained from normal individuals and from psoriatic patients. In the latter case, biopsies were taken in uninvolved skin, in the center of lesions, and at the edge of evolving plaques (spanning uninvolved and involved skin). Alterations in epidermal differentiation were assessed by the distribution of filagrin, involucrin, and epidermal membrane-bound transglutaminase. Capillary leakiness was evaluated by the abundance of plasma proteins such as albumin, fibrinogen, and immunoglobulin G within the epidermis. Typical alterations of epidermal differentiation were already obvious at the edge of the lesions, in areas devoid of vessel abnormalities and leakiness, or significant cellular infiltration. These results strongly suggest that, during the formation of a psoriatic plaque, defects in keratinocyte differentiation precede the development of vascular anomalies.

Laminin provides a better substrate than fibronectin for attachment, growth, and differentiation of 1003 embryonal carcinoma cells.

Culture of cells in hormonally defined media has allowed (a) the demonstration of physiological responses from cells usually unable to express them in vitro and (b) the study of the effects on growth and differentiation of diffusible factors and attachment factors. The embryonal carcinoma line 1003 forms multidifferentiated tumors in vivo but is unable to differentiate in vitro when grown in serum-containing medium. In a defined medium containing insulin, transferrin, selenium, and fibronectin as attachment factors, 1003 cells grow for several generations and differentiate into neurons and embryonic mesenchyme (Darmon et al., 1981, Dev. Biol. 85: 463-473). In the present work the effects of fibronectin and laminin were compared. In the presence of laminin the cells attached and spread better, grew faster, and could be plated at lower densities. Neurite extension was also better under these conditions and most importantly, it was found that laminin induced an important formation of muscular tissue when the cells had been seeded at low densities. Multinucleated myotubes could be stained with antibodies directed against embryonic muscular myosin. Coating the dishes with polylysine or adding FGF or serum-spreading factor to the medium allowed growth of low-density cultures with fibronectin instead of laminin but muscular differentiation was not detected under these conditions. Addition of fibronectin to laminin-containing medium did not inhibit muscular differentiation.

Sequence of a cDNA encoding human keratin No 10 selected according to structural homologies of keratins and their tissue-specific expression.

We present here the nucleotide sequence of a 1700 bp-long cDNA encoding human epidermal keratin No. 10 (56.5 kDa). cDNA clones of the acidic keratin family were first isolated from a pBR322 human epidermal cDNA library by hybridization with a probe coding for keratin No. 14. Differential hybridization using total cDNA probes prepared from poly(A)+ RNA extracted either from epidermis (which contains keratin No. 10) and from squamous carcinoma or hepatoma cell lines (which do not express keratin No. 10) made possible the selection of clones potentially coding for keratin No. 10. The 1.7 kb sequence exhibits the characteristic features of an acidic keratin with a constant central rod domain and C-terminal variable structures. Moreover, the sequence shows extensive homologies with the cDNA of murine keratin No. 10.

Modulation of HPV18 and BPV1 transcription in human keratinocytes by simian virus 40 large T antigen and adenovirus type 5 E1A antigen.

Transcription of early open reading frames initiated from the long control region (LCR) of HPV18 and BPV1 is known to be modulated by homologous and heterologous papillomarvirus E2 gene products. Using CAT constructs transfected into normal human keratinocytes, we show that SV40 large T antigen activates transcription from the LCR of both viruses, whereas Ad5-E1a antigen represses transcription from the HPV18-LCR but activates transcription from BPV1-LCR. Experiments using constructs containing subfragments of the HPV18-LCR cloned in enhancer configuration ahead of the SV40 early promoter or the HSV1-Tk promoter suggest that the effect of Ad5-E1a antigen on HPV18 transcription is probably due to a repression of the enhancer function of the LCR. The mechanism of transcription stimulation by SV40 large T antigen is less clear. The 230 bp Rsa1-Rsa1 central domain of the HPV18-LCR seems involved both in transcriptional stimulation by SV40 large T antigen and transcriptional inhibition by adenovirus E1a antigen.