To our knowledge, the present study is the first systematic analysis of regulation by TGFβ on gene expression of its own receptors and Smads, in human articular chondrocytes. Our study shows that TGFβ exerts a differential effect on the transcription of genes implicated in the canonical Smads pathway. While TGFβ upregulates its receptors and Smad3 for short incubation (at least at mRNA level), it downregulates them in the long term. In addition, it upregulates Smad7 and does not significantly alter Smad2 and Smad4 expression. This positive and negative feedback loop of the TGFβ pathway induces differential response of chondrocytes to TGFβ. The mechanisms responsible for modulation of Smads and for TGFβ receptor expression seem to be different. Indeed, TGFβ downregulates both receptors, at least by modifying the mRNA stability. This process appears slowly (after 24 hours of treatment). On the contrary, TGFβ1 quickly regulates Smad3 and Smad7 mRNA levels by a mechanism independent of mRNA stability.
Our results suggest that following TGFβ1 administration a rapid activation of TGFβ signalling occurs, characterised by phosphorylation of Smad2/3 and upregulation of TβRI, TβRII and Smad3 (at least at mRNA level). Thereafter, a negative feedback loop of the TGFβ1 signalling pathway occurs with a decline of these receptors and R-Smad expression and a simultaneous rise in the inhibitory Smad7 level. The activation of P-Smad2/3 and upregulation of Smad7 after 30 minutes of TGFβ treatment are consistent with observations from Jimenez's group obtained with human and bovine chondrocytes .
The downregulation of TGFβ receptors by its own ligand is controversial, and is dependent on cell type as well as on duration of TGFβ1 incubation. In lung fibroblasts, TGFβ1 induced an increased type I receptor expression by enhancing the transcription of this gene , whereas its expression is not modulated or downregulated in osteoblasts [17, 18]. Similarly, TβRII can be downregulated or upregulated by its own ligand [18–20]. In addition, in osteoblasts TGFβ1 reduces the amount of specific TβRII at the cell surface but does not affect the mRNA steady-state level .
We have established that, in human OA chondrocytes, TGFβ acts, at least in part, by strongly decreasing the mRNA stability of its receptors. This rapid turnover potentially allows the receptor rate to change rapidly in response to its own ligand. We cannot, however, exclude the possibility that TGFβ downregulates its receptors also at the transcriptional and translational levels.
Concerning Smad effectors, our results are consistent with data obtained in normal skin fibroblasts  - which demonstrated that TGFβ treatment causes an upregulation of antagonistic Smad7, and a dramatic decrease in Smad3 mRNA expression. Interestingly, the mRNA level of the closely related Smad2 was not affected by 48 hours of treatment with TGFβ1. A differential regulation between R-Smads has already been described in lung epithelial and mesangial cells [23, 24] and may lead to a variation in the cell response according to the level of TGFβ. Similar to findings obtained in fibroblasts  or in mesangial cells , we established that the downregulation of Smad3 mRNA expression in TGFβ-treated chondrocytes was not due to decreased transcript stability, suggesting a transcriptional effect of TGFβ. Further experiments, such as nuclear run-on or gene reporter assays, would be required to definitively state this hypothesis.
In contrast to Smad3, Smad7 mRNA expression was rapidly and markedly induced by TGFβ. These findings are agreement with reports describing Smad7 as an immediate-early gene target of TGFβ in MV1Lu cells, HaCaT cells  and skin fibroblasts . Increased expression of the inhibitor Smad7 has been associated with inhibition of TGFβ signalling. Smad7 could negatively regulate TGFβ signalling; on one hand by inhibiting R-Smad activation by TβRI or by enhancing TβRI degradation in the cytoplasm, and on the other hand by disrupting the formation of the TGFβ-induced functional Smad-DNA complex in the nucleus .
These TGFβ-induced modifications on expression of TGF receptors and Smads may participate in the chondrocyte-phenotype changes observed in OA, a pathology associated, at least in the first stage, with an increase in the TGFβ level . Modifications of Smad3 expression are associated with OA [6, 7], and its expression stimulates type II collagen synthesis caused by TGFβ1 . Moreover, activation of Smad pathways by transfection with a dominant-negative Smad7 retroviral vector or constitutively active TβRII abolished retinoic acid-induced inhibition of chondrogenesis, suggesting that TGFβ receptor/Smad signalling is essential for this process . Furthermore, ectopic expression of TβRII restores TGFβ sensitivity and increases aggrecan and col2 expression, in IL1-treated or passaged chondrocytes, respectively ( and unpublished personal data).
Our experiments indicate that TGFβ1 exerts a differential effect on profiling of gene expression in chondrocytes according to the duration of treatment. A short TGFβ1 administration (1 hour) induces Sox9 expression, followed, after 3 hours, by induction of collagen type II expression. This effect was transient, but a second peak of collagen II expression appears after 24 hours of incubation of TGFβ1. These data suggest that at least two different mechanisms are responsible for cell response to TGFβ. A short TGFβ administration may activate the Smad2/3 pathway (upregulation of TβRI, TβRII and Smad3, and phosphorylation of Smad2/3), leading to an increase of Sox9, which, in turn, may induce collagen type II expression. Thereafter, a negative feedback loop occurs, characterised by a reduction of TβRI, TβRII and Smad3 expression and simultaneous induction of the inhibitory Smad7. This feedback leads to blockage of Smad2/3-mediated TGFβ signalling and reduction of Sox9, and furthermore to reduced collagen type II expression.
On the contrary, longer incubation leads an additional response to TGFβ but with a different pattern of matrix gene expression. This late response is associated with increased atypical collagen expression (COL1A1 and COL10A1) and reduction of aggrecan expression. These data suggest that a noncanonical pathway could be involved in this late response to TGFβ. Several pathways may be implied. In particular, the reduction of TβRI expression may change the ratio between TβRI and ALK1, another type I TGFβ receptor recently identified in chondrocytes, favouring TGFβ signalling via the Smad1/5/8 route and, subsequently, chondrocyte terminal differentiation [28, 29].
Finally, in the present report we show that Sp1 is involved in the regulation of TGFβ receptors and cell response to TGFβ. TGFβ acts controversially on Sp1 expression. Previous data obtained in rabbit chondrocytes showed that TGFβ decreases Sp1 expression and binding activity , whereas recent studies indicate that TGFβ induces Sp1 in skin fibroblasts . Our data show that Sp1 is downregulated in human chondrocytes, suggesting that this negative effect does not depend on the species but is cell-type specific.
The mechanism by which TGFβ regulates Sp1 expression is still unclear. In particular, the role of Smads in the regulation of Sp1 promoter activity is not known. Analysis of the Sp1 promoter (region -2,000 to +1) with Patch_Search , however, shows numerous putative binding sites for Smad3 and Smad4 in the 1,000 base pair upstream transcription initiation site of the Sp1 gene. An extensive study will be required to determine whether Smads directly or indirectly regulate Sp1 expression. Besides, a recent study shows that Smads bind in association with Sp1 to the CC(GG)-rich TGFβ1 responsive element of the human α1 type I collagen promoter that lacks the classical Smad recognition element, thus enhancing the binding of Sp1 and in this manner activating the collagen promoter . Numerous studies indicate also that Sp1 cooperate with Smads to regulate the expression of TGFβ target genes [3, 31, 34, 35].
Importantly, restoration by Sp1 of TGFβ receptor expression after inhibition by TGFβ1 strongly suggests that inhibition of Sp1 by TGFβ is a potential cause of TGFβ-mediated suppression. These results were in agreement with previous reports that demonstrate Sp1 is a transactivator of both TGFβ receptors [36, 37]. Moreover, a key role of Sp1 in the Smad7 induction by TGFβ was recently established in pancreatic cancer cells . In our study, however, Sp1 does not regulate Smad7 expression, suggesting that the regulatory mechanism of Smad7 is cell specific.
Interestingly, Sp1 ectopic expression permits one to maintain, even after 24 hours of treatment, the early cell response to TGFβ (induction of Sox9, COL2A1) and to counteract the late response (upregulation of COL1A1, COL10A1, repression of aggrecan). These data suggest that targeting Sp1 expression in association to TGFβ treatment might be an innovative strategy to maintain or induce the chondrocyte phenotype.