Abstract
IntroductionTransforming growth factor beta (TGFβ) plays a central role in morphogenesis, growth, and cell differentiation. This cytokine is particularly important in cartilage where it regulates cell proliferation and extracellular matrix synthesis. While the action of TGFβ on chondrocyte metabolism has been extensively catalogued, the modulation of specific genes that function as mediators of TGFβ signalling is poorly defined. In the current study, elements of the Smad component of the TGFβ intracellular signalling system and TGFβ receptors were characterised in human chondrocytes upon TGFβ1 treatment.MethodsHuman articular chondrocytes were incubated with TGFβ1. Then, mRNA and protein levels of TGFβ receptors and Smads were analysed by RT-PCR and western blot analysis. The role of specific protein 1 (Sp1) was investigated by gain and loss of function (inhibitor, siRNA, expression vector).ResultsWe showed that TGFβ1 regulates mRNA levels of its own receptors, and of Smad3 and Smad7. It modulates TGFβ receptors post-transcriptionally by affecting their mRNA stability, but does not change the Smad-3 and Smad-7 mRNA half-life span, suggesting a potential transcriptional effect on these genes. Moreover, the transcriptional factor Sp1, which is downregulated by TGFβ1, is involved in the repression of both TGFβ receptors but not in the modulation of Smad3 and Smad7. Interestingly, Sp1 ectopic expression permitted also to maintain a similar expression pattern to early response to TGFβ at 24 hours of treatment. It restored the induction of Sox9 and COL2A1 and blocked the late response (repression of aggrecan, induction of COL1A1 and COL10A1).ConclusionsThese data help to better understand the negative feedback loop in the TGFβ signalling system, and enlighten an interesting role of Sp1 to regulate TGFβ response.
Highlights
Transforming growth factor beta (TGFb) plays a central role in morphogenesis, growth, and cell differentiation
transforming growth factor beta 1 (TGFb1) downregulates TGFb receptors and Smad3, and upregulates Smad7 We investigated the effect of TGFb1 on mRNA expression of TGFb signalling genes in a dose-dependent manner, using real-time reverse transcriptase (RT)-polymerase chain reaction (PCR) (Figure 1)
A 48-hour incubation with TGFb1 significantly reduced the expression of both TGFb receptors and Smad3, whereas the Smad7 mRNA level was increased
Summary
Transforming growth factor beta (TGFb) plays a central role in morphogenesis, growth, and cell differentiation. This cytokine is important in cartilage where it regulates cell proliferation and extracellular matrix synthesis. Transforming growth factor beta (TGFb) controls a wide range of cellular responses, including differentiation, cell proliferation, migration, apoptosis, extracellular matrix remodelling and development. Following binding of TGFb to TbRII, the ligand-bound type II receptor forms an oligomeric complex with the type I receptor, resulting in TbRI phosphorylation. In contrast to Smad2/3, Smad forms a stable association with the receptor complex and prevents receptormediated phosphorylation of pathway-restricted Smads, resulting in disruption of TGFb signalling [4]
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