Effects of interleukin-11 on the expression of human bone sialoprotein gene

Interleukin-11 (IL-11) is a bone marrow stromal fibroblast-derived cytokine with a wide spectrum of activities in different biological systems. IL-11 and IL-6 are two cytokines known to rely on osteoblast-osteoclast communication for their effects on osteoclast differentiation. Bone sialoprotein (BSP) is a mineralized connective tissue-specific protein expressed in differentiated osteoblasts, odontoblasts, and cementoblasts. To determine the molecular basis of the transcriptional regulation of the human BSP gene by IL-11, we conducted real-time polymerase chain reactions (PCR), transient transfection analyses with chimeric constructs of the human BSP gene promoter linked to a luciferase reporter gene, gel mobility shift assays, and a chromatin immunoprecipitation assay using human osteoblast-like Saos2 cells. IL-11 (20 ng/ml) increased BSP, Runx2, and Osterix mRNA levels at 6 h and the alkaline phosphatase (ALP) mRNA level at 12 h in osteoblast-like Saos2 cells. In a transient transfection assay, IL-11 (20 ng/ml, 12 h) increased luciferase activities of constructs between -60LUC and -868LUC including the human BSP gene promoter. Transcriptional stimulations by IL-11 were partially inhibited in the constructs that included 2-bp mutations in the cAMP response element 1 (CRE1, -72 to -79) and CRE2 (-667 to -674). When mutations were made in pairs of CRE1 and CRE2 in -868LUC, the effect of IL-11 on luciferase activity was almost totally abrogated. Transcriptional activities induced by IL-11 were inhibited by protein kinase A, tyrosine kinase, ERK1/2, and PI3-kinase inhibitors. Gel mobility shift analyses showed that IL-11 increased nuclear proteins binding to CRE1 and CRE2. CREB1, phospho-CREB1, c-Fos, and c-Jun antibodies disrupted the formation of CRE1 and CRE2 protein complexes. These data demonstrate that IL-11 stimulates BSP gene transcription via CRE1 and CRE2 elements in the human BSP gene promoter.