title: Protamine stimulates bone sialoprotein gene expression.
abstract: Protamine is a small, arginine-rich, nuclear protein that replaces histone late in the haploid phase of spermatogenesis and is believed to be essential for sperm head condensation and DNA stabilization. Protamine has many biological activities and has roles in hematopoiesis, immune responses, the nervous system and bone metabolism. Bone sialoprotein (BSP) is a mineralized connective tissue-specific protein expressed in differentiated osteoblasts that appears to function in the initial mineralization of bone. Protamine (71.35 ng/ml) increased BSP mRNA levels by 6h in osteoblast-like ROS 17/2.8 cells. In a transient transfection assay, protamine (71.35 ng/ml) increased luciferase activity of the construct (-116 to +60) in ROS 17/2.8 cells and rat bone marrow stromal cells. Luciferase activities induced by protamine were blocked by protein kinase A, tyrosine kinase and ERK1/2 inhibitors. Introduction of 2 bp mutations to the luciferase constructs showed that the effects of protamine were mediated by a cAMP response element (CRE), a fibroblast growth factor 2 response element (FRE) and a homeodomain protein-binding site (HOX). Gel shift analyses showed that protamine (71.35 ng/ml) increased the nuclear protein binding to CRE, FRE and HOX. CREB, phospho-CREB, c-Fos, c-Jun, JunD and Fra2 antibodies disrupted the formation of CRE-protein complexes. Dlx5, Msx2, Runx2 and Smad1 antibodies disrupted FRE- and HOX-protein complex formations. These studies demonstrate that protamine induces BSP transcription by targeting CRE, FRE and HOX sites in the proximal promoter of the rat BSP gene. Moreover, phospho-CREB, c-Fos, c-Jun, JunD, Fra2, Dlx5, Msx2, Runx2 and Smadl transcription factors appear to be key regulators of protamine effects on BSP transcription.
authors.affiliation: Department of Periodontology, Nihon University School of Dentistry at Matsudo, Chiba, 271-8587, Japan.
authors.forename: Liming
authors.initials: L
authors.lastname: Zhou
authors.forename: Hiroyoshi
authors.initials: H
authors.lastname: Matsumura
authors.forename: Masaru
authors.initials: M
authors.lastname: Mezawa
authors.forename: Hideki
authors.initials: H
authors.lastname: Takai
authors.forename: Yohei
authors.initials: Y
authors.lastname: Nakayama
authors.forename: Makoto
authors.initials: M
authors.lastname: Mitarai
authors.forename: Yorimasa
authors.initials: Y
authors.lastname: Ogata
mesh_terms: D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D047369:Chromatin Immunoprecipitation; D050659:Core Binding Factor Alpha 1 Subunit / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects*; D017930:Genes, Reporter; D058574:Integrin-Binding Sialoprotein / Q000235:genetics* / Q000378:metabolism; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011479:Protamines / Q000494:pharmacology*; D051381:Rats; D014162:Transfection
chemical_list: D050659:Core Binding Factor Alpha 1 Subunit; D058574:Integrin-Binding Sialoprotein; D011479:Protamines; C493717:Runx2 protein, rat; D008156:Luciferases