Update on peptidylarginine deiminases and deimination in skin physiology and severe human diseases.

Deimination (or citrullination) is a recently described post-translational modification, but its consequences are not yet well understood. It is catalysed by peptidylarginine deiminases (PADs). These enzymes transform arginyl residues involved in a peptidyl link into citrullyl residues in a calcium-dependent manner. Several PAD substrates have already been identified like filaggrin and keratins K1 and K10 in the epidermis, trichohyalin in hair follicles, but also ubiquitous proteins like histones. PADs act in a large panel of physiological functions as cellular differentiation or gene regulation. It has been suggested that deimination plays a role in many major diseases such as rheumatoid arthritis, multiple sclerosis, Alzheimer's disease and psoriasis. Five human genes (PADIs), encoding five highly conserved paralogous enzymes (PAD1-4 and 6), have been characterized. These genes are clustered in a single locus, at 1p35-36 in man. Only PAD1-3 are expressed in human epidermis. PADs seem to be controlled at transcriptional, translational and activity levels and they present particular substrate specificities. In this review, we shall discuss these main biochemical, genetic and functional aspects of PADs together with their pathophysiological implications.

A family based study shows no association between rheumatoid arthritis and the PADI4 gene in a white French population.

BACKGROUND: Autoantibodies to citrullinated proteins (ACPA) are considered a specific marker for rheumatoid arthritis. Peptidylarginine deiminase (PAD) is the enzyme that converts arginyl into citrullyl residues; different isoforms of the enzyme are expressed in mammals. It has been suggested that the PADI4 gene may contribute to genetic susceptibility to rheumatoid arthritis, but conflicting results have been obtained in different populations. OBJECTIVE: To test the hypothesis that the PADI4 gene may confer susceptibility to rheumatoid arthritis in a white French population, using powerful and highly reliable family based association tests. METHODS: DNA samples were analysed from 100 families where one member was affected by rheumatoid arthritis and both parents were available for sampling. Five single nucleotide polymorphisms, located within the PADI4 gene and in its close proximity, were genotyped by restriction fragment length polymorphism, and haplotypes were constructed. The analysis involved use of the transmission disequilibrium test and genotype relative risk. ACPA were detected by ELISA on cyclic citrullinated peptides and on human deiminated fibrinogen. RESULTS: No single SNP or haplotype was associated with the disease, or was preferentially transmitted. No association was found when patients were partitioned according to ACPA positivity. CONCLUSIONS: No PADI4 haplotype is associated with rheumatoid arthritis in a white French population. The role of genes encoding the other PAD isoforms, or modulating tissue expression or enzyme activity, remains to be elucidated.

Corneodesmosin expression in psoriasis vulgaris differs from normal skin and other inflammatory skin disorders.

SUMMARY: Corneodesmosin (Cdsn) is a late differentiation epidermal glycoprotein putatively involved in keratinocyte adhesion. The Cdsn gene lies within the susceptibility region on chromosome 6p21.3 (PSORS1) for psoriasis, a common chronic disfiguring skin disease. A particular allelic variant of Cdsn has a strong association with psoriasis. Therefore, genetically and biologically, Cdsn is a possible candidate gene for psoriasis susceptibility. To investigate a potential role for Cdsn in psoriasis pathogenesis, protein expression studies were performed by quantitative immunohistochemistry on normal skin, psoriatic skin (lesional and nonlesional), and other skin disorders using monoclonal antibodies (G36-19 and F28-27). In normal and nonlesional skin, Cdsn was expressed in stratum corneum and one or two layers of superficial stratum granulosum. In lesional psoriasis, there was a significant increase in Cdsn expression, which was observed in multiple layers of stratum spinosum and in stratum corneum. The expression pattern varied from granular, cytoplasmic immunoreactivity to cell surface labeling with weakly immunoreactive cytoplasm. In chronic atopic dermatitis, lichen planus, mycosis fungoides, and pityriasis rubra pilaris, Cdsn immunoreactivity was confined to stratum corneum and upper stratum granulosum with no stratum spinosum immunoreactivity. Immunoelectron microscopy of normal and lesional psoriatic skin demonstrated Cdsn release concomitant with involucrin incorporation into cell envelopes and completed before mature envelope formation. Extracellular release of Cdsn occurred at a lower level of the epidermis in psoriasis than normal skin. These protein expression studies provide evidence of altered Cdsn expression in psoriasis consistent with a role of Cdsn in disease pathogenesis. Further functional and genetic studies of Cdsn are justified to determine its role as a potential psoriasis-susceptibility factor.

Refined characterization of corneodesmosin proteolysis during terminal differentiation of human epidermis and its relationship to desquamation.

Corneodesmosin is a putative adhesion glycoprotein located in the extracellular part of the desmosomes in the upper layers of the epidermis. Synthesized by granular keratinocytes as a 52-56-kDa protein, corneodesmosin is progressively proteolysed during corneocyte maturation. This processing is a prerequisite for desquamation. Two glycine- and serine-rich domains of the protein might take on the conformation of adhesive secondary structures similar to glycine loops. Corneodesmosin proteolysis was further characterized. Deglycosylation experiments and reactivity with lectins demonstrated that the corneodesmosin carbohydrate moiety does not prevent the proteolysis. Immunoblotting, immunohistochemistry, and immunoelectron microscopy experiments using affinity-purified anti-peptide antibodies raised to four of the five structural domains of corneodesmosin and a monoclonal antibody against its fifth central domain showed that the first step in corneodesmosin processing is the cleavage of its extremities and probably occurs before its incorporation into desmosomes. Then the glycine loop-related domains are cleaved, first the N-terminal and then part of the C-terminal domain. At the epidermis surface, the multistep proteolytic cleavage leaves intact only the central domain, which was detected on exfoliated corneocytes and probably lacks adhesive properties. Importantly, corneodesmosin was demonstrated to be a preferred substrate of two serine proteases involved in desquamation, the stratum corneum tryptic and chymotryptic enzymes.

Identification of six novel polymorphisms in the human corneodesmosin gene.

Psoriatic epidermis is characterised by a defective differentiation program leading to an abnormal permeability barrier and impaired desquamation. The corneodesmosin gene (CDSN) or "S" gene is a strong candidate in psoriasis susceptibility, due first to its genomic position ("S" gene, 160 kb telomeric to HLA-C) and second to its expression and function in the epidermis. Moreover, an association between CDSN and psoriasis vulgaris was recently shown in Caucasian populations. In order to pursue the CDSN polymorphism analysis, we determined the sequence of its alleles in 14 HLA-Cw6-positive individuals. A 4.6 kb genomic fragment encompassing the first exon, the unique intron and the coding sequence of the second exon was amplified from 8 psoriatic patients and 6 controls. Allelic discrimination was performed by restriction fragment length polymorphism analysis. The entire coding sequence and the intron boundaries of 27 alleles were sequenced. A total of 26 dimorphic sites were found, 23 consisting in single nucleotide polymorphisms (SNPs) and 3 in triplet modifications. Five out of the 23 SNPs have not been previously reported, and among them, one causes amino-acid exchange leading to the suppression of a potential chymotrypsin site. Among the triplet modifications, one leads to deletion of one out of five consecutive valines in the protein. The high polymorphism of the gene allowed the identification of 13 different alleles. These haplotypes will permit additional family-based studies that could provide new genetic support for the involvement of CDSN in psoriasis susceptibility. Moreover, the establishment of an extensive catalogue of CDSN alleles will allow functional analyses of the different protein isoforms.

Interconnections between E2-dependent regulation of cell cycle progression and apoptosis in MCF-7 tumors growing on nude mice.

Growth of human breast adenocarcinoma MCF-7 cells as a tumor on nude mice is dependent on estrogen. It has been shown that estrogen withdrawal (EW) induces a partial regression of the tumor via an inhibition of cell proliferation and an induction of apoptosis. We investigated in this in vivo model the underlying molecular mechanisms of the hormone-dependent regulation of cell cycle machinery and apoptosis. We found that, 2 days after EW, the tumor protein levels of p21 rose, whereas those of Rb proteins decreased in parallel with the decrease in the proportion of tumor cells in S phase and the increase of the tumor apoptotic index. Between 3 and 7 days after EW, apoptosis was inhibited and tumor proliferation returned to the control value. There was a concomitant decline in p21 and an elevation of Rb tumor protein content. Slight variations of cyclin D protein level were observed in MCF-7 tumors over the time course following EW treatment. Bcl-2 overexpression not only inhibited apoptosis induced by EW but also modulated hormone-dependent cell cycle regulation. First, the analysis of phosphorylation status of Rb protein and the measurement of the proportion of tumor cells in S phase indicated that Bcl-2 overexpression results in a decrease of DNA synthesis induced by estradiol. Furthermore, after EW, Bcl-2-induced inhibition of hormone-dependent apoptosis was associated with an inhibition of Rb protein downregulation, a sustained level of p21 protein, and a prolonged inhibition of cell cycle progression. These results suggest that, in human hormone-dependent breast cancers, cross-talk exists between the signaling pathways which lead to regulation of cell cycle progression and apoptosis.

Expression cloning of human corneodesmosin proves its identity with the product of the S gene and allows improved characterization of its processing during keratinocyte differentiation.

In human epidermis and other cornified squamous epithelia, corneodesmosin is located in the desmosomes of the upper living layers, and in related structures of the cornified layers, the corneodesmosomes. During maturation of the cornified layers, the protein undergoes a series of cleavages, thought to be a prerequisite of desquamation. Partial amino acid sequencing of corneodesmosin fragments suggested that it is related to the product of the S gene, previously identified in the human major histocompatibility complex. We report the expression cloning of corneodesmosin cDNA from a human epidermis library screened with monoclonal antibodies. Sequencing demonstrated that corneodesmosin is really the product of the S gene. However, analysis of 20 alleles of the gene revealed that its product is 27 amino acids longer than initially reported. Two additional polymorphic sites were described, and the position of the unique intron was ascertained. Corneodesmosin cDNA expression in COS-7 cells led to secretion of the protein. Precise epitope mapping allowed further characterization of the molecular forms of corneodesmosin present in the most superficial cornified layers, where fragments corresponding to the central region of the protein were detected. This indicated a cleavage of the N- and C-terminal domains of corneodesmosin before desquamation. These serine- and glycine-rich domains are proposed to mediate an adhesive function.

Corneodesmosin, a corneodesmosome-specific basic protein, is expressed in the cornified epithelia of the pig, guinea pig, rat, and mouse.

Proteolysis of corneodesmosin, a 52- to 56-kDa basic protein located in the extracellular part of the modified desmosomes (corneodesmosomes) of human cornified epithelia, is thought to be a key event of desquamation. Three monoclonal antibodies specific for human corneodesmosin were used to search for the expression of the protein in other mammals. Cryosections of pig, guinea pig, rat, and mouse cornified tissues and proteins sequentially extracted from the corresponding epithelia were analyzed by immunofluorescence and immunoblotting, respectively. Two of the antibodies (F28-27 and B17-21) showed, on the epidermis of the four species and on the cornified epithelia of the rat tongue and esophagus, the same labeling as on human epidermis. Cytoplasmic in the lower granular layer, then pericellular microgranular, the labeling progressively disappeared in the lower cornified layer. By contrast, it persisted up to the surface in the rat tail epidermis. The two antibodies immunodetected basic proteins extracted with isotonic buffer from the epidermis of the pig (50 kDa), guinea pig (52 kDa), and mouse (75 kDa) and from the cornified epithelia of the rat (75 kDa). Immunoreactive proteins of lower Mr were also extracted partly with urea and partly with a reducing agent. The third antibody (G36-19) presented the same reactivities except on murine tissues, where it was unreactive. Our results show that the location, the biochemical characteristics, and the processing of corneodesmosin are similar in five mammals, including humans, suggesting an important role for this protein. They open the way to studies of its function in desquamation using various animal models.

Overexpression of vascular endothelial growth factor induces cell transformation in cooperation with fibroblast growth factor 2.

Vascular endothelial growth factor (VEGF) is a family of homodimeric proteins produced from a single gene by alternative splicing of the VEGF transcript. VEGF induces in vivo angiogenesis and vascular permeability. We have recently demonstrated that VEGF is an autocrine growth factor for retinal pigment epithelial (RPE) cells. To further understand the role of VEGF, we overexpressed VEGF in rat RPE cells. The transfected cells exhibited a growth advantage in vitro and an increased response to the mitogenic effect of fibroblasts growth factor-2 (FGF-2), and formed colonies in soft agar upon FGF-2 addition. Moreover, analysis of FGF-receptors evidenced a dramatic increase in FGFR-1 mRNA and protein level, supporting the hypothesis that this receptor mediates the transforming effect of FGF-2. These results reveal that the oncogenic role of VEGF is exerted through a cross regulation between VEGF and FGF signal transduction pathways.

Characterization and purification of human corneodesmosin, an epidermal basic glycoprotein associated with corneocyte-specific modified desmosomes.

Using monoclonal antibodies, we identified a new protein in mammalian epidermis, which we called corneodesmosin. It is located in the extracellular part of the modified desmosomes in the cornified layer of the tissue, and its proteolysis (from 52-56 to 33 kDa) is thought to be a major prerequisite of desquamation. We have now further characterized human corneodesmosin. Proteolysis of purified cornified cell envelopes produced immunoreactive fragments, confirming the covalent linkage of the protein to these structures. Sequential extraction of epidermal proteins indicated that the 52-56-kDa precursor form of the protein exists in two distinct pools, one extracted with a nondenaturing hypotonic buffer, and the other with urea. Two-dimensional gel analysis and reactivity with phosphoserine-specific antibodies showed that it is a basic phosphoprotein. Deglycosylation experiments, reactivity with lectins, and chromatography on concanavalin A-Sepharose indicated that corneodesmosin is N-glycosylated. Partial sequences, 10 and 15 amino acids long, of the purified 52-56-kDa corneodesmosin showed identity with sequences predicted from a previously cloned gene, proved to be expressed in the epidermis and designated S. This indicates that corneodesmosin is probably encoded by the S gene, the function of which was unknown until now. A model of corneodesmosin maturation during cornification is proposed.

Transforming growth factor beta 1-induced apoptosis in human ovarian carcinoma cells: protection by the antioxidant N-acetylcysteine and bcl-2.

We have previously shown that transforming growth factor beta 1 (TGF-beta 1) inhibits growth and induces apoptosis in NIH-OVCAR-3 ovarian adenocarcinoma cells. In this study, we investigated the role of the reactive oxygen species in the TGF-beta 1 signaling pathways. We found that both TGF-beta 1 and an oxidant, hydrogen peroxide, rapidly increase the expression of c-fos and c-jun genes and induce cell death by apoptosis; these effects are inhibited by the antioxidant N-acetylcysteine. In contrast, N-acetylcysteine did not influence TGF-beta 1-mediated cell cycle arrest at the G1-S transition. TGF-beta 1 down-regulated the endogenous expression of the anti-apoptotic bcl-2 gene, and overexpression of this gene inhibited TGF-beta 1-induced apoptosis. Taken together, these results suggest that TGF-beta 1 activates multiple signaling pathways in the NIH-OVCAR-3 cell line and that the reactive oxygen species play a role in the early gene responses and apoptosis induced by TGF-beta 1.

Vasculotropin/vascular endothelial growth factor is an autocrine growth factor for human retinal pigment epithelial cells cultured in vitro.

Vasculotropin (VAS), also called vascular endothelial growth factor (VEGF) or vascular permeability factor, is a secreted growth factor whose target cell specificity has been reported as restricted to vascular endothelium. Its effects are mediated by at least two distinct membrane-spanning tyrosine kinase receptors, KDR and flt-1; the expression of which also seems restricted to vascular endothelium. We describe here that cultured human retinal pigment epithelial (HRPE) cells express both KDR and flt-1 receptors, bind VAS/VEGF on two high affinity sites (apparent Kd of 9 and 210 pM corresponding to 940 and 18,800 sites per cell) and proliferate or migrate upon recombinant VAS/VEGF addition. HRPE cells also express the mRNA corresponding to the 121 and 165 amino acid forms of VAS/VEGF. HRPE cells release in their own culture medium and store in their extracellular matrix self-mitogenic and chemoattractant factors indistinguishable from 121 and 165 VAS/VEGF isoforms. The autocrine role of VAS/VEGF was confirmed by the inhibition of these bioactivities by neutralizing specific anti-VAS/VEGF antibodies.

Early gene responses associated with transforming growth factor-beta 1 growth inhibition and autoinduction in MCF-7 breast adenocarcinoma cells.

In the human breast carcinoma cell line (MCF-7), exogenous TGF-beta 1 induces a dose-dependent inhibition of cell proliferation. In a MCF-7 cell subline [MCF-7(-)], which has an undetectable level of type II TGF-beta receptor, exogenous TGF-beta 1 does not inhibit cell proliferation but is still able to induce its own message. In both cell lines, TGF-beta 1 stimulates expression of c-jun, whereas a rapid, transient and marked increase in c-fos mRNA is only observed in the MCF-7 cells sensitive to the growth inhibitory effect of TGF-beta 1. Depletion of protein kinase C abolishes the c-fos but not the c-jun response to TGF-beta 1. Our results suggest that growth inhibition and autoinduction by TGF-beta 1 are mediated by different signalling pathways. In addition, a PKC-dependent increase in c-fos expression seems to be associated with the growth inhibitory effect of TGF-beta 1.

Vasculotropin-VEGF stimulates retinal capillary endothelial cells through an autocrine pathway.

PURPOSE: To determine whether bovine retinal endothelial cells (BRECs) bind, synthesize, and respond to vasculotropin-vascular endothelial growth factor (VAS-VEGF). METHODS: Cultured BRECs were tested for their ability to bind 125I VAS-VEGF and their response to the growth and migration-promoting effect of VAS-VEGF. Total RNAs extracted from BRECs were reverse transcribed and amplified by polymerase chain reaction using VAS-VEGF primers. The translation was assessed by a Western blot analysis and a radioreceptor assay in the BREC-conditioned medium. Neutralization with anti-VAS-VEGF antibodies ascertained the autocrine role of VAS-VEGF. RESULTS: BRECs bind VAS-VEGF on two high-affinity binding sites (apparent Kd of 2 and 56 pM) and can proliferate and migrate upon the addition of recombinant VAS-VEGF. Furthermore, BRECs synthesize and secrete into their own culture medium a mitogen related to VAS-VEGF as far as two factors are concerned: chromatographic behavior on heparin-affinity columns, and cross-reactivity with recombinant VAS-VEGF to the binding to its receptors or antibodies. Neutralization of the purified conditioned medium with anti-VAS-VEGF antibodies revealed that VAS-VEGF can act on BRECs through an autocrine pathway. CONCLUSIONS: This is the first description of an autocrine regulation of endothelial cell growth by VAS-VEGF that could be involved in the pathogenesis of retinal neovascularization.

Transforming growth factor beta 1 (TGF-beta 1) inhibits growth of a human ovarian carcinoma cell line (OVCCR1) and is expressed in human ovarian tumors.

The effects of EGF and TGF-beta 1 on the proliferation of 2 ovarian carcinoma cell lines (IGROV1 and OVCCR1) were evaluated. The cell lines were adapted to grow in a restricted serum (0.5%) medium. EGF was required for proliferation of both ovarian cell lines. Low doses of TGF-beta 1 inhibited clonogenic capacity and attenuated the EGF-mediated stimulation of DNA synthesis in OVCCR1 cells. TGF-beta 1 inhibited OVCCR1 cell proliferation by blocking the cell cycle at the G1/S transition. TGF-beta 1 did not affect either clonal or monolayer growth of IGROV1 cells. Both cell lines express type-I and type-III TGF-beta receptors, suggesting that the unresponsiveness of IGROV1 cells to TGF-beta 1 occurs at a post-receptor level. TGF-beta 1 mRNA was detected in OVCCR1 cells and in 8 out of 11 of the ovarian tumor specimens examined.

Antiproliferative effect of phorbol esters on MCF-7 human breast adenocarcinoma cells: relationship with enhanced expression of transforming growth-factor-beta 1.

In human breast carcinoma MCF-7 cells, phorbol diesters inhibit proliferation and induce cell maturation. We have recently reported that exogenous TGF-beta 1 reverses the resistance of a breast adenocarcinoma MCF-7 subline (MCF-7:RPh-4) to these phorbol ester effects. Here, we investigated the involvement of TGF-beta 1 in the PKC-mediated inhibition of breast-cancer cell proliferation. Parental MCF-7-conditioned medium contained a 20-fold higher transforming activity on NRK-49F fibroblasts than the TPA-resistant subline. TPA increased TGF-beta activity in MCF-7 conditioned medium. MCF-7 cells also expressed more TGF-beta 1 mRNA than the resistant subline. TPA induced a dose-dependent increase in TGF-beta 1 mRNA levels that paralleled the inhibitory effect on MCF-7 proliferation. The lower level of TGF-beta mRNA expression in TPA resistant subline was not modified after addition of TPA, but was significantly increased in the presence of exogenous TGF-beta 1. These data argue in favor of a role of endogenous TGF-beta 1 in the maturation process induced by protein kinase C activation.

[Regulation by protein kinase C of TGF-beta 1 expression in cultured cells of breast adenocarcinoma]. / Régulation par la protéine kinase C de l'expression du TGF-beta 1 dans les cellules d'adénocarcinome mammaire en culture.

In human breast carcinoma MCF-7 cells, phorbol diesters inhibit proliferation and induce cell maturation. We investigated the involvement of TGF-beta 1 in the PCK-mediated inhibition of breast cancer cell proliferation. Using an RNase protection assay, we showed that TPA induced a dose-dependent increase in levels of TGF-beta 1 mRNA that paralleled the inhibitory effect on MCF-7 proliferation. Similar results were obtained with another TPA-sensitive breast cancer cell line (BT-20). TPA did not increase TGF-beta 1 mRNA levels in the MCF-7:RPh-4 and T47D cell lines, which are both insensitive to the growth inhibitory effects of phorbol esters. In addition, the increase in TGF-beta 1 mRNA level was not observed after treatment of the MCF-7 cell with other inducers of cell differentiation such as forskolin, DMF, HMBA and sodium butyrate. The induction of TGF-beta 1 mRNA by TPA along with its inhibitory effect on cell proliferation suggests that TGF-beta 1 mediates, at least in part, the inhibitory effect of PKC activation.

Transforming growth factor beta (TGF-beta) reverses phorbol diester resistance of a breast adenocarcinoma (MCF-7) subline.

We investigated the effects of TGF-beta on a MCF-7 subline (MCF-7:RPh-4) which is resistant to phorbol diesters with respect to growth inhibition and estrogen receptor content modulation. This biological unresponsiveness of MCF-7:RPh-4 cells to phorbol esters seems to be unrelated to activation of protein kinase C. In the presence of 80 nM PMA (12-O-tetradecanoylphorbol-13-acetate), TGF-beta induced a dose-dependent inhibition of MCF-7:RPh-4 cell proliferation. MCF-7:RPh-4 cells grown in PMA-free medium for at least 28 days remained insensitive to PMA but lost sensitivity to TGF-beta. Under these conditions, addition of 80 nM PMA restored sensitivity to TGF-beta. In the presence of a fixed concentration of TGF-beta, the dose-dependent inhibition of proliferation and the decrease in estrogen receptor content induced by PMA were comparable to those observed in PMA-treated parental MCF-7 cells. These observations indicate that TGF-beta reverses PMA resistance in MCF-7:RPh-4 cells. In addition, TGF-beta does not modify the basal or PMA-stimulated phosphorylation of Mr 28,000 endogenous protein. These results suggest that TGF-beta interferes with the protein kinase C pathway independently of enzyme activation.