Sp1 family of transcription factors regulates the human alpha2 (XI) collagen gene (COL11A2) in Saos-2 osteoblastic cells.

UNLABELLED: Genes encoding type XI collagen, normally associated with chondrogenesis, are also expressed by osteoblasts. By studying Saos-2 cells, we showed that the transcription factors, Sp1, Sp3, and Sp7 (Osterix), regulate COL11A2 expression through its proximal promoter. The findings indicate both ubiquitous and osteoblast-specific mechanisms of collagen gene regulation. INTRODUCTION: Type XI collagen is essential for skeletal morphogenesis. Collagen XI gene regulation has been studied in chondrocytes but not in osteoblasts. MATERIALS AND METHODS: We cultured Saos-2 cells, a human osteosarcoma-derived line of osteoblasts, and analyzed them for alpha2(XI) protein and COL11A2 regulatory mechanisms. RESULTS AND CONCLUSIONS: Although types I and V were the dominant collagens deposited by Saos-2 cells, they expressed COL11A2 mRNA, and alpha2(XI) chains were present in the extracellular matrix. The COL11A2 promoter region (from -149 to -40) containing three Sp1 binding sites was required for promoter activity in transient transfection assays. All three Sp1 sites were critical for binding by nuclear proteins in electrophoretic mobility shift assays. Further analysis using consensus oligonucleotides and specific antibodies as well as chromatin immunoprecipitation assay implicated Sp1 and Sp3 in binding to this promoter region. Overexpressing Sp1 or Sp3 significantly increased COL11A2 promoter activity and endogenous COL11A2 gene expression, an effect that was suppressed by the Sp1-binding inhibitor mithramycin A. Further experiments showed that Sp1, Sp3, CREB-binding protein (CBP), p300, and histone deacetylase (HDAC) were physically associated and HDAC inhibitors (trichostatin A or NaB) upregulated COL11A2 promoter activity and endogenous gene expression. Another Sp1 family member, Sp7 (Osterix), was expressed in Saos-2 cells, but not in chondrocytes, and was shown by chromatin immunoprecipitation to occupy the COL11A2 promoter. Overexpressing Sp7 increased COL11A2 promoter activity and endogenous gene expression, an effect also blocked by mithramycin A. Using siRNA to knockdown Sp1, Sp3, or Sp7, it was shown that depression of any of them decreased COL11A2 promoter activity and endogenous gene expression. Finally, primary cultures of osteoblasts expressed COL11A2 and Sp7, upregulated COL11A2 promoter activity and endogenous gene expression when Sp1, Sp3, or Sp7 were overexpressed, and downregulated them when Sp1, Sp3, or Sp7 were selectively depressed. The results establish that Sp1 proteins regulate COL11A2 transcription by binding to its proximal promoter and directly interacting with CBP, p300, and HDAC.

Regulation of ionomycin-mediated granule release from rat basophil leukemia cells.

Cross-linking the high affinity IgE receptor on the rat basophil leukemia clone 2H3 (RBL-2H3) cell line, an vitro model for mast cell signaling, results in granule release. A great deal of research has focused on the earliest steps in this signaling cascade resulting in models which include the participation of lyn, syk, phospholipase C (PLC), protein kinase C (PKC) and intracellular calcium mobilization. In an effort to look at pathways downstream of calcium mobilization, ionomycin-mediated granule release was studied. The kinase inhibitors PP1 (src family), GF109203X (PKC), PD98059 (MEK1/2), and U0126 (MEK1/2) substantially inhibited ionomycin-mediated granule release, while the p38 kinase inhibitor SB203580 did not. Both p38 and erk were phosphorylated upon ionomycin treatment, but only extracellular regulated kinase (erk) activation was completely inhibited by PP1 treatment and partially inhibited by the MEK inhibitors, thus, correlating with the granule release data. Interestingly, while GF109203X alone had no affect on erk activation, combining it with U0126 completely blocked this response. This suggests the existence an alternate pathway for erk activation that is MEK independent and PKC dependent. Experiments in which ionomycin and PP1 were titrated (independently) demonstrated a correlation between erk phosphorylation and granule release, implicating erk in a PP1-inhibitable pathway operating downstream of calcium and controlling mast cell degranulation.

Fusion of green fluorescent protein to the C-terminus of granulysin alters its intracellular localization in comparison to the native molecule.

The engineering of green fluorescent protein (GFP) fusion constructs in order to visibly tag a protein of interest has become a commonly used cell biology technique. Although caveats to this approach are obvious, literature reports in which the chimeric molecule behaves differently than the native molecule are scant. This brief report describes one such case. Granulysin, a small lytic and antimicrobial protein produced by cytotoxic lymphocytes, traffics to the regulated secretory system and is subsequently released from cells upon proper stimulus. In an attempt to elucidate mechanisms by which it accumulates in and is released from cytolytic granules, GFP was fused to the C-terminus of granulysin and expressed in an NK cell line. A control construct expressing the native protein was similarly expressed. The data demonstrate that, while the fusion protein is expressed and secreted, its subcellular localization is altered in comparison to native granulysin. Thus, the addition of GFP to the C-terminus of granulysin obscures the signal(s) that cytotoxic lymphocytes use to sort it to the regulated secretory pathway despite its normal biosynthesis and secretion. This example is offered as a cautionary account for other researchers who contemplate using this technology.