Sp1 Is Required for the Early Response of α2(I) Collagen to Transforming Growth Factor-β1

Abstract

It is currently debated whether AP1 or Sp1 is the factor that mediates transforming growth factor ␤1 (TGF-␤) stimulation of the human ␣2(I) collagen (COL1A2) gene by binding to an upstream promoter element (TbRE). The present study was designed to resolve this controversy by correlating expression of COL1A2, AP1, and Sp1 in the same cell line and under different experimental conditions. The results strongly indicate that Sp1 is required for the immediate early response of COL1A2 to TGF-␤ and AP1 is not. The Sp1 inhibitor mithramycin blocked stimulation of ␣2(I) collagen mRNA accumulation by TGF-␤, whereas the AP1 inhibitor curcumin had no effect. Furthermore, antibodies against Jun-B and c-Jun failed to identify immunologically related proteins in the TbRE-bound complex, irrespective of whether they were purified from untreated or TGF-␤-treated cells. AP1 did bind to the TbRE probe in vitro, but only in the absence of the upstream Sp1 recognition sequence. Based on this finding and DNA transfection results, we conclude that the AP1 sequence of the TbRE represents a cryptic site used under experimental conditions that either eliminate the more favorable Sp1 binding site or force the balance toward the less probable. Finally, a combination of cell transfections and DNA-binding assays excluded that COL1A2 transactivation involves the retinoblastoma gene product (pRb), an activator of Sp1, the pRb-related protein p107, an inhibitor of Sp1, or the Sp1-related repressor, Sp3.

Sp1 and Smad Proteins Cooperate to Mediate Transforming Growth Factor-β1-induced α2(I) Collagen Expression in Human Glomerular Mesangial Cells

“…1B). These results are consistent with previous findings from other groups using human fibroblasts (32,50). The inhibitory effect of mithramycin A was specific since transcription of other genes such as TIMP-1 was not blocked by this chemical antagonist (Fig.…”

“…The next day, they were preincubated with mithramycin A (100 mM) for 17 h or curcumin (20 M) for 30 min before addition of 1 ng/ml TGF-␤1 or control vehicle for 24 h. These conditions are similar to the ones showing efficient inhibition of Sp1 binding by mithramycin A and of AP-1 activation by curcumin (32). Total RNA was analyzed by Northern blot as described previously (36).…”

“…Doublestranded oligonucleotides were labeled with [␥- 32 P]ATP and T4 polynucleotide kinase. Five g of nuclear extracts were incubated for 30 min at 4°C with 40,000 cpm of 32 P probe in 10 mM Hepes, pH 7.9, 5% glycerol, 0.1 mM EDTA, 1 mM dithiothreitol, and 1 g of poly(dI-dC)⅐poly(dI-dC).…”

Role of TGF-β1 and JNK signaling in capillary tube patterning

“…A hallmark for the potency of TGF-␤ to induce diverse cellular functions is the ability of the TGF-␤-activated signaling intermediates, the SMADs, to cooperate with each other and with other members of DNA binding proteins from many different families to elicit specific transcriptional responses in a given cellular context (29). The transcription factor Sp1 has been shown to mediate the transcriptional activation of collagen expression by TGF-␤ (22,62). To determine whether transcriptional regulation was involved in the bipolarized capillary tube patterning observed, the DNA intercalator WP631, which has been shown to inhibit collagen expression (19), was used.…”

Cbfa1 Contributes to the Osteoblast-specific Expression of type I collagen Genes

“…The ␣1(I) and ␣2(I) chains are encoded by two distinct genes that are expressed most highly in two cell types: the fibroblast and the osteoblast. Moreover, the expression of these two genes is often regulated by identical transcription factors (3)(4)(5)(6)(7). The type I collagen genes are expressed in osteoblastic cells at all stages during development and throughout life (8), suggesting that the factor(s) controlling their expression in these cells could also control osteoblast differentiation and function.…”