The immediate early gene product hCYR61 localizes to the secretory pathwayin human osteoblasts

The human cysteine-rich protein 61 (hCYR61) belongs to the growing CCN (CYR 61/CTGF/NOV) family of immediate early genes, which modulate cell growth an d differentiation. hCYR61 is regulated by 1 alpha,25-dihydroxyvitamin D-3 ( 1,25-(OH)(2)D-3) and growth factors in fetal human osteoblasts (hFOB cells) . The murine homologue CYR61 was characterized as an extracellular matrix-a ssociated protein that modulates basic fibroblast growth factor signaling, angiogenesis, and binds to integrin alpha(v)beta(3). Here we report the int racellular localization of the human CYR61 gene product by overexpressing f usion proteins with green fluorescent protein (GFP) in primary osteoblasts and the hFOB cell line. Full-length hCYR61-GFP localizes to the Golgi appar atus and cytoplasmatic granules, indicating targeting to the secretory path way. Secretion of hCYR61 from osteoblasts is verified by Western blot detec tion from cellular supernatants, A truncated hCYR61-GFP fusion protein cont aining only the 34 N-terminal amino acids of hCYR61 also localizes to the G olgi apparatus mainly in the perinuclear region, which suggests that the N- terminus of hCYR61 is sufficient to target the protein to the secretory pat hway. In summary, our results present the first evidence that human CYR61 l ocalizes to the secretory pathway in primary osteoblasts and hFOB cells, an d that it is secreted from these cells. The N-terminal 34 amino acids of hC YR61 provide a sufficient Golgi targeting sequence. Together with the immed iate early regulation of hCYR61 mRNA by 1,25-(OH)(2)D-3, this suggests that hCYR61 might function as an extracellular signaling molecule in human bone . (Bone 27:53-60; 2000) (C) 2000 by Elsevier Science Inc. All rights reserv ed.