Randomized phase II selection trial of FLASH and conventional radiotherapy for patients with localized cutaneous squamous cell carcinoma or basal cell carcinoma: A study protocol.

Background: Cutaneous basal cell carcinoma (BCC) and squamous cell carcinoma (SCC) are the most prevalent skin cancers in western countries. Surgery is the standard of care for these cancers and conventional external radiotherapy (CONV-RT) with conventional dose rate (0.03-0.06 Gy/sec) represents a good alternative when the patients or tumors are not amenable to surgery but routinely generates skin side effects. Low energy electron FLASH radiotherapy (FLASH-RT) is a new form of radiotherapy exploiting the biological advantage of the FLASH effect, which consists in delivering radiation dose in milliseconds instead of minutes in CONV-RT. In pre-clinical studies, when compared to CONV-RT, FLASH-RT induced a robust, reproducible and remarkable sparing of the normal healthy tissues, while the efficacy on tumors was preserved. In this context, we aim to prospectively evaluate FLASH-RT versus CONV-RT with regards to toxicity and oncological outcome in localized cutaneous BCC and SCC. Methods: This is a randomized selection, non-comparative, phase II study of curative FLASH-RT versus CONV-RT in patients with T1-T2 N0 M0 cutaneous BCC and SCC. Patients will be randomly allocated to low energy electron FLASH-RT (dose rate: 220-270 Gy/s) or to CONV-RT arm. Small lesions (T1) will receive a single dose of 22 Gy and large lesions (T2) will receive 30 Gy in 5 fractions of 6 Gy over two weeks.The primary endpoint evaluates safety at 6 weeks after RT through grade ≥ 3 toxicity and efficacy through local control rate at 12 months. Approximately 60 patients in total will be randomized, considering on average 1-2 lesions and a maximum of 3 lesions per patients corresponding to the total of 96 lesions required. FLASH-RT will be performed using the Mobetron® (IntraOp, USA) with high dose rate functionality.LANCE (NCT05724875) is the first randomized trial evaluating FLASH-RT and CONV-RT in a curative setting.

Skin Colonization by Staphylococcus aureus Precedes the Clinical Diagnosis of Atopic Dermatitis in Infancy.

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.

Activin a inhibits antigen-induced allergy in murine epicutaneous sensitization.

Activin A, a member of the TGFß superfamily, is involved in physiological processes such as cell differentiation, tissue homeostasis, wound healing, reproduction, and in pathological conditions, such as fibrosis, cancer, and asthma. Activin enhances mast cell maturation, as well as regulatory T-cell and Langerhans cell differentiation. In this study we investigated the potential role of activin in epicutaneous sensitization with ovalbumin (OVA), notably with respect to its effect on known Th2-polarization. For this purpose, transgenic mice overexpressing activin in keratinocytes and their wild-type (WT) controls were sensitized epicutaneously with OVA. Skin biopsies were analyzed with regard to histopathological features and mRNA expression of pro-inflammatory and Th1/Th2 cytokines, and Ig levels were measured in the serum. Unexpectedly, activin overexpressing animals were protected from Th2-cytokine expression and induction of OVA-specific IgE levels compared to WT animals. On the other hand, transgenic mice were more susceptible to inflammation compared to WT littermates after tape-stripping and saline (vehicle) or OVA application, as shown by increased pro-inflammatory cytokine mRNA levels and neutrophil accumulation at the site of the treatment. We conclude that activin protects from antigen-induced cutaneous Th2-polarization through modulation of the immune response. These findings highlight the role of activin in cutaneous sensitization, allergy, and in skin homeostasis.

Spontaneous atopic dermatitis-like symptoms in a/a ma ft/ma ft/J flaky tail mice appear early after birth.

Loss-of-function mutations in human profilaggrin gene have been identified as the cause of ichthyosis vulgaris (IV), and as a major predisposition factor for atopic dermatitis (AD). Similarly, flaky tail (a/a ma ft/ma ft/J) mice were described as a model for IV, and shown to be predisposed to eczema. The aim of this study was to correlate the flaky tail mouse phenotype with human IV and AD, in order to dissect early molecular events leading to atopic dermatitis in mice and men, suffering from filaggrin deficiency. Thus, 5-days old flaky tail pups were analyzed histologically, expression of cytokines was measured in skin and signaling pathways were investigated by protein analysis. Human biopsies of IV and AD patients were analyzed histologically and by real time PCR assays. Our data show acanthosis and hyperproliferation in flaky tail epidermis, associated with increased IL1ß and thymic stromal lymphopoietin (TSLP) expression, and Th2-polarization. Consequently, NFκB and Stat pathways were activated, and IL6 mRNA levels were increased. Further, quantitative analysis of late epidermal differentiation markers revealed increased Small proline-rich protein 2A (Sprr2a) synthesis. Th2-polarization and Sprr2a increase may result from high TSLP expression, as shown after analysis of 5-days old K14-TSLP tg mouse skin biopsies. Our findings in the flaky tail mouse correlate with data obtained from patient biopsies of AD, but not IV. We propose that proinflammatory cytokines are responsible for acanthosis in flaky tail epidermis, and together with the Th2-derived cytokines lead to morphological changes. Accordingly, the a/a ma ft/ma ft/J mouse model can be used as an appropriate model to study early AD onset associated with profilaggrin deficiency.

The human epidermal differentiation complex: cornified envelope precursors, S100 proteins and the 'fused genes' family.

  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.

Sox9/Sox6 and Sp1 are involved in the insulin-like growth factor-I-mediated upregulation of human type II collagen gene expression in articular chondrocytes.

Type II collagen is a marker of articular cartilage encoded by the COL2A1 gene. The nature of the trans factors involved in the upregulation of this gene by insulin-like growth factor-I (IGF-I) remains unclear. We found that IGF-I increased type II collagen synthesis by a transcriptional control mechanism involving a 715-bp region within the COL2A1 first-intron specific enhancer. The overproduction of L-Sox5/Sox6/Sox9 and Sp1 and decoy experiments targeting these factors demonstrated their action in concert in IGF-I trans-activation. These results were supported by the data obtained in knockdown experiments in which siRNA against Sox9/Sox6 and Sp1 prevented the IGF-I-induced increase in collagen II production. Indeed, each of these trans-activators increased the expression of others. IGF-I increased the binding of Sox9 and Sp1/Sp3 to their cis elements in the enhancer, and we provide the first evidence of Sox9 interaction with the promoter by chromatin immunoprecipitation. Interactions with COL2A1 were also observed for Sp1, p300/CBP, and Tip60. Finally, a physical interaction between Sox9, p300, Sp3, and Sp1 was detected. These data demonstrate the role of Sox9, Sp1/Sp3, and euchromatin-associated factors (p300, Tip60) in the IGF-I-induced upregulation of COL2A1, indicating possible use of this growth factor in articular cartilage engineering applications to promote repair in patients with degenerative diseases, such as osteoarthritis.

The p65 subunit of NF-κB inhibits COL1A1 gene transcription in human dermal and scleroderma fibroblasts through its recruitment on promoter by protein interaction with transcriptional activators (c-Krox, Sp1, and Sp3).

Transcriptional mechanisms regulating type I collagen genes expression in physiopathological situations are not completely known. In this study, we have investigated the role of nuclear factor-κB (NF-κB) transcription factor on type I collagen expression in adult normal human (ANF) and scleroderma (SF) fibroblasts. We demonstrated that NF-κB, a master transcription factor playing a major role in immune response/apoptosis, down-regulates COL1A1 expression by a transcriptional control involving the -112/-61 bp sequence. This 51-bp region mediates the action of two zinc fingers, Sp1 (specific protein-1) and Sp3, acting as trans-activators of type I collagen expression in ANF and SF. Knockdown of each one of these trans factors by siRNA confirmed the trans-activating effect of Sp1/Sp3 and the p65 subunit of NF-κB trans-inhibiting effect on COL1A1 expression. Despite no existing κB consensus sequence in the COL1A1 promoter, we found that Sp1/Sp3/c-Krox and NF-κB bind and/or are recruited on the proximal promoter in chromatin immunoprecipitation (ChIP) assays. Attempts to elucidate whether interactions between Sp1/Sp3/c-Krox and p65 are necessary to mediate the NF-κB inhibitory effect on COL1A1 in ANF and SF were carried out; in this regard, immunoprecipitation assays revealed that they interact, and this was validated by re-ChIP. Finally, the knockdown of Sp1/Sp3/c-Krox prevents the p65 inhibitory effect on COL1A1 transcription in ANF, whereas only the siRNAs targeting Sp3 and c-Krox provoked the same effect in SF, suggesting that particular interactions are characteristic of the scleroderma phenotype. In conclusion, our findings highlight a new mechanism for COL1A1 transcriptional regulation by NF-κB, and these data could allow the development of new antifibrotic strategies.

Characterization of a non-fibrillar-related collagen in the mollusc Haliotis tuberculata and its biological activity on human dermal fibroblasts.

In invertebrates, members of the collagen family have been found in various phyla. Surprisingly, in mollusc, little is known about such molecules. In this study, we characterize the full-length abalone type IV collagen and we analysed its biological effects on human fibroblast in order to gain insights about this molecule in molluscs and particularly clues about its roles. We screened a cDNA library of Haliotis tuberculata hemocytes. The expression pattern of the transcript is determined using real-time polymerase chain reaction and in situ hybridization. The close identity between α1(IV) C-terminal domain and the vertebrate homologue led us to produce, purify and test in vitro a recombinant protein corresponding to this region using human dermal fibroblasts cell culture. The biological effects were evaluated on proliferation and on differentiation. We found that the 5,334-bp open reading frame transcript encodes a protein of 1,777 amino acids, including an interrupted 1,502-residue collagenous domain and a 232-residue C-terminal non-collagenous domain. The expression pattern of this transcript is mainly found in the mantle and hemocytes. The recombinant protein corresponding α1(IV) C-terminal domain increased fibroblast proliferation by 69% and doubled collagen synthesis produced in primary cultures. This work provides the first complete primary structure of a mollusc non-fibrillar collagen chain and the biological effects of its C-terminal domain on human cells. In this study, we prove that the NC1 domain from a molluscan collagen can improve human fibroblast proliferation as well as differentiation.

Homeodomain-only protein HOP is a novel modulator of late differentiation in keratinocytes.

The homeodomain-only protein (HOP) contains an atypical homeodomain which is unable to bind to DNA due to mutations in residues important for DNA binding. Recently, HOP was reported to regulate proliferation/differentiation homeostasis in different cell types. In the present study, we performed transcriptional profiling of cultured primary human keratinocytes and noted a robust induction of HOP upon calcium-induced cell differentiation. Immunohistochemistry of human skin localized HOP to the granular layer in the epidermis. Overexpression of HOP using a lentiviral vector up-regulated FLG and LOR expression during keratinocyte differentiation. Conversely, decreasing HOP expression using small interfering RNA markedly reduced the calcium-induced expression of late markers of differentiation in vitro, with the most prominent effect on profilaggrin (FLG) mRNA. Moreover, mRNA levels of profilaggrin and loricrin were downregulated in the epidermis of HOP knockout mice. Analysis of skin disorders revealed altered HOP expression in lichen planus, psoriasis and squamous cell carcinoma (SCC). Our data indicate that HOP is a novel modulator of late terminal differentiation in keratinocytes.

[Pathogenesis of atopic dermatitis]. / Pathogenèse de la dermatite atopique.

Although atopic dermatitis (AD) is a very frequent disease in our society, it is still poorly understood. AD mainly results from a complex interaction between a cutaneous barrier dysfunction, a dysregulation of the immune system and environmental factors. Recent studies have highlighted new mutations in genes coding for skin proteins inducing AD. Furthermore, a new cytokine named TSLP was discovered. TSLP plays a major role in allergic inflammation and represents a big step further in the understanding of AD pathogenesis. However, there are still a lot of unknown factors in this disease, which are actually thouroughly investigated in numerous studies.

Chondroitin sulphate decreases collagen synthesis in normal and scleroderma fibroblasts through a Smad-independent TGF-beta pathway--implication of C-Krox and Sp1.

Despite several investigations, the transcriptional mechanisms which regulate the expression of both type I collagen genes (COL1A1 and COL1A2) in either physiological or pathological situations, such as scleroderma, are not completely known. In this study, we determined the effects of both native ichtyan chondroïtin sulphate (CS) and its derived hydrolytic fragments (CSf) on human normal (NF) and scleroderma (SF) fibroblasts. Here, we demonstrate for the first time that CS and CSf exert an inhibitory effect on type I collagen protein synthesis and decrease the corresponding mRNA steady-state levels of COL1A1 and COL1A2 in NF and SF. These glycosaminoglycan molecules repress COL1A1 gene transcription through a -112/-61 bp sequence upstream the start site of transcription and imply hc-Krox and Sp1 transcription factors. In addition, CS and CSf induced a down-regulation of TbetaRI expression. As a conclusion, our findings highlight a possible new role for CS and CSf as anti-fibrotic molecules and could help in elucidating the mechanisms of action by which CS and CSf exert their inhibitory effect on type I collagen synthesis.

Interleukin-6 (IL-6) and/or soluble IL-6 receptor down-regulation of human type II collagen gene expression in articular chondrocytes requires a decrease of Sp1.Sp3 ratio and of the binding activity of both factors to the COL2A1 promoter.

Type II collagen is composed of alpha1(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 Sp1.Sp3. 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-6.sIL-6R induced an increase in Sp3 recruitment to the detriment of Sp1. Knockdown of Sp1.Sp3 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 Sp1.Sp3 complex is involved. Additionally, Sp1 was shown to interact with Sp3 and HDAC1. Indeed, overexpression of a full-length 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 Sp1.Sp3 ratio and DNA-binding activities, thus inhibiting COL2A1 transcription.

Human collagen Krox up-regulates type I collagen expression in normal and scleroderma fibroblasts through interaction with Sp1 and Sp3 transcription factors.

Despite several investigations, the transcriptional mechanisms that regulate the expression of both type I collagen genes (COL1A1 and COL1A2) in either physiological or pathological situations, such as scleroderma, are not completely known. We have investigated the role of hc-Krox transcription factor on type I collagen expression by human dermal fibroblasts. hc-Krox exerted a stimulating effect on type I collagen protein synthesis and enhanced the corresponding mRNA steady-state levels of COL1A1 and COL1A2 in foreskin fibroblasts (FF), adult normal fibroblasts (ANF), and scleroderma fibroblasts (SF). Forced hc-Krox expression was found to up-regulate COL1A1 transcription through a -112/-61-bp sequence in FF, ANF, and SF. Knockdown of hc-Krox by short interfering RNA and decoy strategies confirmed the transactivating effect of hc-Krox and decreased substantially COL1A1 transcription levels in all fibro-blast types. The -112/-61-bp sequence bound specifically hc-Krox but also Sp1 and CBF. Attempts to elucidate the potential interactions between hc-Krox, Sp1, and Sp3 revealed that all of them co-immunoprecipitate from FF cellular extracts when a c-Krox antibody was used and bind to the COL1A1 promoter in chromatin immunoprecipitation assays. Moreover, hc-Krox DNA binding activity to its COL1A1-responsive element is increased in SF, cells producing higher amounts of type I collagen compared with ANF and FF. These data suggest that the regulation of COL1A1 gene transcription in human dermal fibroblasts involves a complex machinery that implicates at least three transcription proteins, hc-Krox, Sp1, and Sp3, which could act in concert to up-regulate COL1A1 transcriptional activity and provide evidence for a pro-fibrotic role of hc-Krox.

The phylogenetically conserved molluscan chitinase-like protein 1 (Cg-Clp1), homologue of human HC-gp39, stimulates proliferation and regulates synthesis of extracellular matrix components of mammalian chondrocytes.

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.

c-Krox down-regulates the expression of UDP-glucose dehydrogenase in chondrocytes.

Chondrocyte glycosaminoglycan (GAG) synthesis is regulated by the availability of UDP-glucuronate, the substrate of glucuronosyl transferases which form the GAG chains in proteoglycans and hyaluronan. UDP-glucose dehydrogenase (UDPGD) is therefore a key enzyme in the synthesis of UDP-glucuronate from glucose. However, the mechanisms regulating its expression in chondrocytes are not fully understood. We investigated the effect of c-Krox, a zinc-finger transcription factor previously shown to modulate several matrix genes, on the synthesis of GAG and transcriptional activity of several UDPGD gene promoter constructs, using transient transfection and decoy experiments in rabbit articular chondrocytes (RACs). We show that overexpression of c-Krox inhibits radiosulfate incorporation into neosynthesized GAG and that the effect was mediated by a cis-sequence located between +18 and +39bp of the UDPGD gene. Since that sequence can also bind Sp1/Sp3 factors, it is likely that c-Krox acts in concert with these proteins to modulate the UDPGD gene expression in articular chondrocytes.

The zinc finger protein cKrox directs CD4 lineage differentiation during intrathymic T cell positive selection.

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.

Sp1 and Sp3 transcription factors mediate interleukin-1 beta down-regulation of human type II collagen gene expression in articular chondrocytes.

Interleukin-1 beta (IL-1 beta) is a pleiotropic cytokine that was shown to inhibit the biosynthesis of articular cartilage components. Here we demonstrate that IL-1 beta 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 beta 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 beta 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 beta 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 beta down-regulation of that gene, as we found previously for transforming growth factor-beta 1, is mediated by an increase in the Sp3/Sp1 ratio. Moreover, IL-1 beta increased steady-state levels of Sp1 and Sp3 mRNAs, whereas it enhanced Sp3 protein expression and inhibited Sp1 protein biosynthesis. Nevertheless, IL-1 beta 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.

SOX9 exerts a bifunctional effect on type II collagen gene (COL2A1) expression in chondrocytes depending on the differentiation state.

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.