The genetic programs directing CD4 or CD8 T cell differentiation in the thymus remain poorly understood. While analyzing gene expression during intrathymic T cell selection, we found that Zfp67, encoding the zinc finger transcription factor cKrox, was upregulated during the differentiation of CD4(+) but not CD8(+) T cells. Expression of a cKrox transgene impaired CD8 T cell development and caused major histocompatibility complex class I-restricted thymocytes to differentiate into CD4(+) T cells with helper properties rather than into cytotoxic CD8(+) T cells, as normally found. CD4 lineage differentiation mediated by cKrox required its N-terminal BTB (bric-a-brac, tramtrack, broad complex) domain. These findings identify cKrox as a chief CD4 differentiation factor during positive selection.

The skin is essential for survival and protects our body against biological attacks, physical stress, chemical injury, water loss, ultraviolet radiation and immunological impairment. The epidermal barrier constitutes the primordial frontline of this defense established during terminal differentiation. During this complex process proliferating basal keratinocytes become suprabasally mitotically inactive and move through four epidermal layers (basal, spinous, granular and layer, stratum corneum) constantly adapting to the needs of the respective cell layer. As a result, squamous keratinocytes contain polymerized keratin intermediate filament bundles and a water-retaining matrix surrounded by the cross-linked cornified cell envelope (CE) with ceramide lipids attached on the outer surface. These cells are concomitantly insulated by intercellular lipid lamellae and hold together by corneodesmosmes. Many proteins essential for epidermal differentiation are encoded by genes clustered on chromosomal human region 1q21. These genes constitute the 'epidermal differentiation complex' (EDC), which is divided on the basis of common gene and protein structures, in three gene families: (i) CE precursors, (ii) S100A and (iii) S100 fused genes. EDC protein expression is regulated in a gene and tissue-specific manner by a pool of transcription factors. Among them, Klf4, Grhl3 and Arnt are essential, and their deletion in mice is lethal. The importance of the EDC is further reflected by human diseases: FLG mutations are the strongest risk factor for atopic dermatitis (AD) and for AD-associated asthma, and faulty CE formation caused by TG1 deficiency causes life-threatening lamellar ichthyosis. Here, we review the EDC genes and the progress in this field.

Atopic dermatitis (AD) has a well-established association with skin colonization or infection by Staphylococcus aureus, which can exacerbate the disease. However, a causal relationship between specific changes in skin colonization during the first years of life and AD development still remains unclear. In this prospective birth cohort study, we aimed to characterize the association between skin colonization and AD development in 149 white infants with or without a family history of atopy. We assessed infants clinically and collected axillary and antecubital fossa skin swabs for culture-based analysis at birth and at seven time points over the first 2 years of life. We found that at age 3 months, S. aureus was more prevalent on the skin of infants who developed AD later on. S. aureus prevalence was increased on infants' skin at the time of AD onset and also 2 months before it, when compared with age-matched, unaffected infants. Furthermore, at AD onset, infants testing positive for S. aureus were younger than uncolonized subjects. In conclusion, our results suggest that specific changes in early-life skin colonization may actively contribute to clinical AD onset in infancy.

Type II collagen is composed of ␣1(II) chains encoded by the COL2A1 gene. Alteration of this cartilage marker is a common feature of osteoarthritis. Interleukin-6 (IL-6) is a pro-inflammatory cytokine that needs a soluble form of receptor called sIL-6R to exert its effects in some cellular models. In that case, sIL-6R exerts agonistic action. This mechanism can make up for the partial or total absence of membrane-anchored IL-6 receptors in some cell types, such as chondrocytes. Our study shows that IL-6, sIL-6R, or both inhibit type II collagen production by rabbit articular chondrocytes through a transcriptional control. The cytokine and/or sIL-6R repress COL2A1 transcription by a ؊63/؊35 sequence that binds Sp1⅐Sp3. Indeed, IL-6 and/or sIL-6R inhibit Sp1 and Sp3 expression and their binding activity to the 63-bp promoter. In chromatin immunoprecipitation experiments, IL-6⅐sIL-6R induced an increase in Sp3 recruitment to the detriment of Sp1. Knockdown of Sp1⅐Sp3 by small interference RNA and decoy strategies were found to prevent the IL-6-and/or sIL-6R-induced inhibition of COL2A1 transcription, indicating that each of these Sp proteins is required for down-regulation of the target gene and that a heterotypic Sp1⅐Sp3 complex is involved. Additionally, Sp1 was shown to interact with Sp3 and HDAC1. Indeed, overexpression of a fulllength Sp3 cDNA blocked the Sp1 up-regulation of the 63-bp COL2A1 promoter activity, and by itself, inhibits COL2A1 transcription. We can conclude that IL-6, sIL-6R, or both in combination decrease both the Sp1⅐Sp3 ratio and DNA-binding activities, thus inhibiting COL2A1 transcription. Extracellular matrix (ECM)6 of articular cartilage contains tissue-specific macromolecules including types II, IX, and XI collagens and the large aggregating proteoglycan (PG) aggrecan (1). Type II collagen is the major collagen synthesized by chondrocytes in mature articular cartilage. Each ␣1(II) procollagen chain of the triple helix is encoded by the COL2A1 gene, whose transcription is regulated by DNA elements within both the promoter and the first intron regions (2). Thus, several binding sites of the intronic enhancer were shown to interact with transcription factors such as Sox9, L-Sox5, and Sox6 (3, 4), required for cartilage-specific expression of type II collagen during chondrogenesis in vivo (5), as well as with zinc finger transcription factors Sp1, Sp3, and C-Krox (6, 7). The three latter proteins are also able to bind to several binding sites identified in a 266-bp promoter of the human COL2A1 gene (6 -8). Sp1 was shown to be a strong activator of COL2A1 gene expression via the promoter binding sites, whereas Sp3 was found to prevent the Sp1 up-regulation of the COL2A1 promoter activity by binding to the same cis-acting elements (7).In healthy cartilage, chondrocytes maintain steady-state expression of collagens and PGs and are sensitive to a number of growth factors and cytokines that either enhance or reduce type II collagen synthesis. In osteoarthritis and rheumatoid arthritis, structural...

Interleukin-1␤ (IL-1␤) is a pleiotropic cytokine that was shown to inhibit the biosynthesis of articular cartilage components. Here we demonstrate that IL-1␤ inhibits the production of newly synthesized collagens in proliferating rabbit articular chondrocytes and that this effect is accompanied by a decrease in the steady-state levels of type II collagen mRNA. IL-1␤ down-regulates COL2A1 gene transcription through a ؊41/؊33 bp sequence that binds a multimeric complex including Sp1 and Sp3 transcription factors. Specificity of IL-1␤ effects on COL2A1 promoter activity was demonstrated in experiments in which transfection of a wild type ؊50/؉1 sequence of COL2A1 promoter as a decoy oligonucleotide abolished the IL-1␤ inhibition of a ؊63/؉47 COL2A1-mediated transcription. By contrast, transfection of the related oligonucleotide harboring a targeted mutation in the ؊41/؊33 sequence did not modify the negative effect the cytokine. Because we demonstrated previously that Sp1 was a strong activator of COL2A1 gene expression via the ؊63/؉1 promoter region, whereas Sp3 overexpression blocked Sp1-induced promoter activity and inhibited COL2A1 gene transcription, we conclude that IL-1␤ down-regulation of that gene, as we found previously for transforming growth factor-␤1, is mediated by an increase in the Sp3/Sp1 ratio. Moreover, IL-1␤ increased steady-state levels of Sp1 and Sp3 mRNAs, whereas it enhanced Sp3 protein expression and inhibited Sp1 protein biosynthesis. Nevertheless, IL-1␤ decreased the binding activity of both Sp1 and Sp3 to the 63-bp short COL2A1 promoter, suggesting that the cytokine exerts a post-transcriptional regulatory mechanism on Sp1 and Sp3 gene expressions. Altogether, these data indicate that modulation of Sp3/Sp1 ratio in cartilage could be a potential target to prevent or limit the tissue degradation.Articular cartilage is a highly specialized tissue composed of a complex extracellular matrix of proteoglycans, collagens, and noncollagenous glycoproteins. Cartilage collagens include type II as the major form and types VI, IX, and XI as minor components (1). Type II collagen is an homotrimer composed of ␣1(II) chains encoded by the COL2A1 gene. Previous studies have delineated minimal sequences in the first intron of human, mouse, and rat COL2A1 genes which are sufficient to direct chondrocyte-specific expression in cultured chondrocytes and transgenic mice (2-5). Several binding sites of the intronic enhancer sequences were shown to interact with transcription factors that form chondrocyte-specific complexes, such as SOX9, L-SOX5, and SOX6 (6, 7), and also with factors having less tissue-specific expression, such as Sp1, Sp3, and C-KROX (5, 8). Indeed, promoter sequences are also implicated through interaction with the intronic enhancer sequence, for tissuespecific expression during in vivo and in vitro chondrogenesis (7, 9, 10). In a 266-bp promoter of the human COL2A1 gene mediating enhanced transcription activity, we identified several binding sites for Sp1, Sp3, and C-KROX (5, 8, 11). Sp1 w...

Members of chitinase-like proteins (CLPs) have attracted much attention because of their ability to promote cell proliferation in insects (imaginal disc growth factors) and mammals (YKL-40).To gain insights into the molecular processes underlying the physiological control of growth and development in Lophotrochozoa, we report here the cloning and biochemical characterization of the first Lophotrochozoan CLP from the oyster Crassostrea gigas (Cg-Clp1). Gene expression profiles monitored by real time quantitative reverse transcription-PCR in different adult tissues and during development support the involvement of this protein in the control of growth and development in C. gigas. Recombinant Cg-Clp1 demonstrates a strong affinity for chitin but no chitinolytic activity, as was described for the HC-gp39 mammalian homolog. Furthermore, transient expression of Cg-Clp1 in primary cultures of rabbit articular chondrocytes as well as the use of both purified recombinant protein and conditioned medium from Cg-Clp1-expressing rabbit articular chondrocytes established that Cg-Clp1 stimulates cell proliferation and regulates extracellular matrix component synthesis, showing for the first time a possible involvement of a CLP on type II collagen synthesis regulation. These observations together with the fact that Cg-Clp1 gene organization strongly resembles that of its mammalian homologues argue for an early evolutionary origin and a high conservation of this class of proteins at both the structural and functional levels.Growth factors orchestrate growth and development in metazoan organisms. Despite the huge variety of growth factors characterized in vertebrates, only a few have been identified in Protostome lineages and are mainly restricted to the Ecdysozoan model organisms Caenorhabditis elegans and Drosophila melanogaster. Thus, the third branch of bilaterian, named Lophotrochozoa, is an obviously understudied group of animals, since none of the major model organisms presently belong to this clade (1). Recent reports show that Lophotrochozoan animals exhibit biological characteristics that are considered ancestral, or at least less derived from the ancestral state than in other established systems that are known (2). Since bivalve mollusks belong to the Lophotrochozoa, they are good candidates to characterize growth factors and then contribute to our understanding of the evolution of growth and developmental regulations in bilaterian animals. Furthermore, a better knowledge of the molecular control of mollusk physiology may help improve the hatchery production of these economically important animals. Unfortunately, the number of growth factor genes identified so far at the molecular level in bivalve mollusks is extremely limited. In the pacific oyster, Crassostrea gigas, only four members of the transforming growth factor-␤ superfamily and their corresponding receptors have already been described (3-6). This lack of knowledge is above all the consequence of both the paucity of genomic sequence information and the difficu...

As a key transcription factor in cartilage formation, SOX9 is a potent activator of type II collagen expression, a phenotypic marker of articular chondrocytes. This study was designed to determine the potential role of SOX9 on COL2A1 gene transcription during chondrocyte dedifferentiation, a characteristic feature of osteoarthritic cartilage that can be partially mimicked in vitro by subculturing primary chondrocytes. Constructs containing different regions from the promoter and the first intron of human COL2A1 gene were transfected in differentiated (primary) and dedifferentiated (passaged) rabbit articular chondrocytes (RAC), together with an expression vector containing or not the SOX9 cDNA. As expected, low levels of SOX9 overexpression were capable of enhancing COL2A1 gene transcription in both fully differentiated and slightly phenotypically altered chondrocytes, through the specific intronic enhancer. In contrast, when overexpressed at high levels, SOX9 induced an inhibition of COL2A1 gene expression, mediated by the -266 bp promoter region, whatever the differentiation state of chondrocytes. However, in the advanced stages of dedifferentiation, SOX9, independently of its expression level, depressed COL2A1 transcriptional activity through the -63 bp short promoter. Although SOX9 has a crucial role in chondrocyte differentiation, our findings indicate that this factor cannot restore the phenotype of osteoarthritic chondrocytes by itself.

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