ADAMTS1 is up-regulated via the SMAD dependent TGF-β signaling pathway in hepatocellular carcinoma

ADAMTS1 (a disintegrin and metalloproteinase with thrombospondin motifs 1), a member of the ADAMTS family, is a critical molecule due to its dual roles as a potent angiogenesis inhibitor and an aggrecanase. The dysregulation of ADAMTS1 is a common feature in many pathophysiological conditions, making the understanding of its regulation vital. The primary focus of this investigation was to explore the regulatory role of SMAD transcription factors, the central mediators of the TGF-β signaling pathway, on ADAMTS1 gene expression in Hep3B cells.

To assess the impact of SMAD factors, changes in both transcriptional activity and mRNA levels of ADAMTS1 were quantified using co-transfection experiments. Ectopic expression of SMAD2, SMAD3, and SMAD4 was validated by preliminary Western blot and Real-time PCR steps. Co-transfection with SMAD2/SMAD4 resulted in a significant 5-fold increase in ADAMTS1 expression at 24 h, while the SMAD3/SMAD4 complex demonstrated a substantial 3.5-fold increase (with induction sustained up to 72 h). Transcriptional activity was confirmed using promoter fragment and co-transfection analyses performed via the calcium-phosphate precipitation method. Crucially, the Electrophoretic Mobility Shift Assay (EMSA) directly confirmed that SMAD transcription factors bind to the ADAMTS1 promoter region, validating a direct regulation hypothesis.

These comprehensive findings demonstrate that ADAMTS1 gene expression is under the direct transcriptional control of SMAD factors in Hep3B cells, showing strong induction by both SMAD2/4 and SMAD3/4 complexes. This study provides the first mechanistic evidence for this direct regulation, significantly enhancing the understanding of how the TGF-β signaling axis controls ADAMTS1 expression. This insight is highly relevant to its pathological functions in angiogenesis, tissue remodeling, and cancer progression.