Abstract
Thrombospondin-1 (TSP-1), a matricellular protein widely acclaimed to be involved in the inhibition of angiogenesis and tumorigenesis, is synthesized and secreted by many cell types, including osteoblast and cancer cells. TSP-1 is highly upregulated during early stage of osteogenesis, whereas it inhibits terminal osteoblast differentiation. Expression of TSP-1 is downregulated in cancer cells, and its ectopic expression has been shown to restrain tumor growth. Transcriptional regulation of TSP-1 in osteogenesis and cancer is poorly understood; this prompted us to study its regulation by the two key regulators of the aforementioned processes: Runx2 and Runx3. Through a PCR-based cDNA subtraction technique, we identified and cloned a cDNA fragment for mouse TSP-1, whose expression was dramatically upregulated in response to Runx2 expression in mesenchymal stem cells. Moreover, TSP-1 expression was considerably reduced in the lung of Runx2 knockout mouse. On the other hand, TSP-1 gene expression drastically increased at both the transcriptional and translational levels in response to Runx3 expression in B16-F10 melanoma cells. In line with this, Runx2 and Runx3 bound to the TSP-1 promoter and stimulated its activity. Hence, these results provide first line of evidence that TSP-1 is a transcriptional target gene of Runx2 and Runx3.
Highlights
Thrombospondins are extracellular calcium binding glycoproteins that belong to a small family of proteins comprised of five members: TSP-1 to TSP-5
Since TSP-1 expression coincides with the initiation of osteogenic gene transcription, we asked if Runx2 can regulate TSP-1 transcription in osteoblasts
By using the same cDNA fragment (Figure 1a) as the probe in Northern blotting, we confirmed that the expression of TSP-1 in C3H10T1/2 mesenchymal stem cells was dramatically upregulated by Runx2 (Figure 1b)
Summary
Thrombospondins are extracellular calcium binding glycoproteins that belong to a small family of proteins comprised of five members: TSP-1 to TSP-5. TSP-1 is a major constituent of bone matrix proteins that is synthesized and secreted by osteoblasts [18]. TSP-1 has been reported to inhibit bone nodule formation, osteoblast differentiation and endochondral ossification [21,22]. Runx regulates the expression of several bone matrix protein genes in osteoblasts, such as type I collagen, osteopontin, osteocalcin, collagenase 3 and SPARC (Secreted Protein, Acidic, Cysteine-Rich) [31]. TSP-1 is a potent inhibitor of angiogenesis and tumorigenesis; still, the role of Runx in transcriptional regulation of TSP-1 in cancer cells is not studied in detail. We studied the two critical Runx transcription factors, Runx and Runx, in regulating the transcription of TSP-1 in osteoblasts and melanoma cells, respectively
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