Despite major advances in the understanding of the intimate mechanisms of transforming growth factor-β (TGF-β) signaling through the Smad pathway, little progress has been made in the identification of direct target genes. In this report, using cDNA microarrays, we have focussed our attention on the characterization of extracellular matrix-related genes rapidly induced by TGF-β in human dermal fibroblasts and attempted to identify the ones whose up-regulation by TGF-β is Smad-mediated. For a gene to qualify as a direct Smad target, we postulated that it had to meet the following criteria: (1) rapid (30 min) and significant (at least 2-fold) elevation of steady-state mRNA levels upon TGF-β stimulation, (2) activation of the promoter by both exogenous TGF-β and co-transfected Smad3 expression vector, (3) up-regulation of promoter activity by TGF-β blocked by both dominant-negative Smad3 and inhibitory Smad7 expression vectors, and (4) promoter transactivation by TGF-β not possible inSmad3 ^−/− mouse embryo fibroblasts. Using this stringent approach, we have identified COL1A2, COL3A1, COL6A1, COL6A3, and tissue inhibitor of metalloproteases-1 as definite TGF-β/Smad3 targets. Extrapolation of this approach to other extracellular matrix-related gene promoters also identified COL1A1 and COL5A2, but not COL6A2, as novel Smad targets. Together, these results represent a significant step toward the identification of novel, early-induced Smad-dependent TGF-β target genes in fibroblasts.

Transforming growth factor-β and fibrosis

Osteosarcomas are the most frequent primary bone sarcomas, affecting mainly children, adolescents, and young adults, and with a second peak of incidence in elderly individuals. The current therapeutic management, a combined regimen of poly-chemotherapy and surgery, still remains largely insufficient, as patient survival has not improved in recent decades. Osteosarcomas are very heterogeneous tumors, both at the intra- and inter-tumor level, with no identified driver mutation. Consequently, efforts to improve treatments using targeted therapies have faced this lack of specific osteosarcoma targets. Nevertheless, these tumors are inextricably linked to their local microenvironment, composed of bone, stromal, vascular and immune cells and the osteosarcoma microenvironment is now considered to be essential and supportive for growth and dissemination. This review describes the different actors of the osteosarcoma microenvironment and gives an overview of the past, current, and future strategies of therapy targeting this complex ecosystem, with a focus on the role of extracellular vesicles and on the emergence of multi-kinase inhibitors.

Hedgehog (Hh) and transforming growth factor-β (TGF-β) family members are involved in numerous overlapping processes during embryonic development, hair cycle, and cancer. Herein, we show that TGF-β induces the expression of the Hh signaling molecules Gli1 and Gli2 in various human cell types, including normal fibroblasts and keratinocytes, as well as various cancer cell lines. Gli2 induction by TGF-β is rapid, independent from Hh receptor signaling, and requires a functional Smad pathway. Gli1 expression is subsequently activated in a Gli2-dependent manner. In transgenic mice overexpressing TGF-β1 in the skin, Gli1 and Gli2 expression is also elevated and depends on Smad3. In pancreatic adenocarcinoma cell lines resistant to Hh inhibition, pharmacologic blockade of TGF-β signaling leads to repression of cell proliferation accompanied with a reduction in Gli2 expression. We thus identify TGF-β as a potent transcriptional inducer of Gli transcription factors. Targeting the cooperation of Hh and TGF-β signaling may provide new therapeutic opportunities for cancer treatment. [Cancer Res 2007;67(14):6981–6]