Differential regulation of osteoadherin (OSAD) by TGF-b1 and BMP-2 Anders P. Rehn a, * , Alistair M. Chalk b , Mikael Wendel a a Center for Oral Biology, Karolinska Institutet, Box 4064, 141 04 Huddinge, Sweden b Department of Cell and Molecular Biology, Karolinska Institutet, 171 77 Stockholm, Sweden Received 22 August 2006 Available online 30 August 2006 Abstract Osteoadherin (OSAD) is a member of the small leucine rich-repeat proteoglycan (SLRP) family. SLRPs are normally found in extra- cellular matrices, but OSAD is the only member restricted to mineralized tissues. We investigated the promoter region of OSAD by in silico analysis and found that the proximal promoter region contains sites for Smad-3, Smad-4, and AP-1. All are effectors of TGF-b family signalling. We tested sensitivity of the promoter to the two TGF-b family members TGF-b1 and BMP-2. We found TGF-b1 to down regulate OSAD, while BMP-2 up regulates OSAD. As a consequence of how OSAD is regulated by TGF-b1 and BMP-2 and its temporal expression pattern in osteoblasts and bone development, we can conclude OSAD as an early marker for terminally dif- ferentiated matrix producing osteoblasts. Ó 2006 Elsevier Inc. All rights reserved. Keywords: Osteoadherin; TGF-b1; BMP-2; Promoter; Osteoblast; Transcription factor binding site Small leucine-rich repeat proteoglycans (SLRPs) are a family of proteins represented in all extracellular matrices (reviewed in [1]). They have diverse functions, but share the leucine-rich repeat (LRR) consensus motif. Osteoadh- erin (OSAD) is a keratan sulphate proteoglycan and belongs to the SLRP family [2,3], it is found exclusively in mineralized tissues [4,5]. OSAD has high affinity for hydroxyapatite, which is a unique feature of OSAD among the SLRPs probably mediated by the extended C-terminal with roughly 60% acidic residues. OSAD is expressed from early differentiated osteoblasts and peaks late in osteoid formation and at the start of mineral deposition and has been proposed as an organizer of mineral formation. The regulation of osteoblast differentiation and bone mineral formation is complex and involves a variety of fac- tors (for reviews, see [6–8]). One major family of factors important for hard tissue development is the family of transforming growth factor-b (TGF-bs, reviewed in [6]) which includes bone morphogenetic proteins (BMPs) and TGF-b family with the subfamily members 1, 2, and 3. The most investigated and stable isoform is TGF-b1, which acts at many stages from osteoblast precursor maturation to mature osteoblasts and matrix formation. In bone, TGF-b1 is secreted and stored in the synthesized mineral matrix and is subsequently released upon resorption by osteoclasts [9]. Dependent on concentration and stage of maturity of the target cell, TGF-b1 signalling can give rise to different responses of the cells. For instance, TGF-b1 stimulates osteoblast progenitor proliferation, expanding the pool of committed osteoblasts and thereby promoting bone matrix production. Conversely, TGF-b1 inhibits later stages of differentiation and mineralization, but inhibits apoptosis of osteoblasts converting into osteocytes [10–13]. TGF-bs signal through a dual receptor system, in which the activated intracellular kinase domains activate either the Smad pathway or MAP-kinase signalling cascade (for extensive reviews on TGF-b signalling, see [6,14]). Smads form complexes with other Smads or transcription factors as they translocate to the nucleus and bind DNA, where they can stimulate or inhibit transcription. The MAPK cascade leads to the nuclear translocation of an activator 0006-291X/$ - see front matter Ó 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.08.133 * Corresponding author. Fax: +46 8 779 31 66. E-mail address: anders.rehn@ki.se (A.P. Rehn). www.elsevier.com/locate/ybbrc Biochemical and Biophysical Research Communications 349 (2006) 1057–1064 BBRC