Abstract
Osteoclasts resorb bone through the formation of a unique attachment structure called the sealing zone. In this study, a role for thyroid hormone receptor-interacting protein 6 (TRIP6) in sealing zone formation and osteoclast activity was examined. TRIP6 was shown to reside in the sealing zone through its association with tropomyosin 4, an actin-binding protein that regulates sealing dimensions and bone resorptive capacity. Suppression of TRIP6 in mature osteoclasts by RNA interference altered sealing zone dimensions and inhibited bone resorption, whereas overexpression of TRIP6 increased the sealing zone perimeter and enhanced bone resorption. Treatment of osteoclasts with lysophosphatidic acid (LPA), which phosphorylates TRIP6 at tyrosine 55 through a c-Src-dependent mechanism, caused increased association of TRIP6 with the sealing zone, as did overexpression of a TRIP6 cDNA bearing a phosphomimetic mutation at tyrosine 55. Further, LPA treatment caused increases in osteoclast fusion, sealing zone perimeter, and bone resorptive capacity. In contrast, overexpression of TRIP6 containing a nonphosphorylatable amino acid residue at position 55 severely diminished sealing zone formation and bone resorption and suppressed the effects of LPA on the cytoskeleton. LPA effects were mediated through its receptor isoform LPA(2), as indicated by treatments with receptor-specific agonists and antagonists. Thus, these studies suggest that TRIP6 is a critical downstream regulator of c-Src signaling and that its phosphorylation is permissive for its presence in the sealing zone where it plays a positive role in osteoclast bone resorptive capacity.
Highlights
Finger domains of ϳ50 – 60 amino acids that function in protein-protein interaction and have been found to be involved in regulating transcription and cytoskeletal organization [2, 3]
These studies suggest that thyroid hormone receptor-interacting protein 6 (TRIP6) is a critical downstream regulator of c-Src signaling and that its phosphorylation is permissive for its presence in the sealing zone where it plays a positive role in osteoclast bone resorptive capacity
Utilizing small interfering RNA knockdowns and overexpression of TRIP6 combined with immunocytochemistry and resorption assays, we found that c-Src regulates osteoclast adhesion and resorption in part through tyrosine phosphorylation of TRIP6
Summary
Finger domains of ϳ50 – 60 amino acids that function in protein-protein interaction and have been found to be involved in regulating transcription and cytoskeletal organization [2, 3]. Podosomes and sealing zones are distinct but structurally related adhesion structures that contain a multitude of actin regulatory proteins and signaling molecules, including the nonreceptor tyrosine kinase c-Src [11]. Multinucleated osteoclasts were generated in the absence of c-Src, these cells lacked the ability to form sealing zones and resorb bone [12,13,14]. Of relevance to this study, c-Src has been shown to phosphorylate tyrosine 55 of TRIP6 in NIH 3T3 cells when stimulated with lysophosphatidic acid [15]. We showed that osteoclasts express multiple isoforms of tropomyosins, actin-binding proteins that stabilize F-actin, and found that tropomyosin 4 (Tm-4) regulates both sealing zone and podosome height [24, 25]. Utilizing small interfering RNA knockdowns and overexpression of TRIP6 combined with immunocytochemistry and resorption assays, we found that c-Src regulates osteoclast adhesion and resorption in part through tyrosine phosphorylation of TRIP6
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