Spp24 Derivatives Stimulate a G_i-Protein Coupled Receptor-Erk1/2 Signaling Pathway and Modulate Gene Expressions in W-20-17 Cells

Abstract

Secreted phosphoprotein 24 kDa (Spp24) is an apatite- and BMP/TGF-β cytokine-binding phosphoprotein found in serum and many tissues, including bone. N-terminally intact degradation products ranging in size from 14 kDa to 23 kDa have been found in bone. The cleavage sites in Spp24 that produce these short forms have not been definitively identified, and the biological activities and mechanisms of action of Spp24 and its degradation products have not been fully elucidated. We found that the C-terminus of Spp24 is labile to proteolysis by furin, kallikrein, lactoferrin, and trypsin, indicating that both extracellular and intracellular proteolytic events could account for the generation of biologically-active Spp18, Spp16, and Spp14. We determined the effects of these truncation products on kinase-mediated signal transduction, gene expression, and osteoblastic differentiation in W-20-17 bone marrow stromal cells cultured in basal or pro-osteogenic media. After culturing for five days, all forms inhibited BMP-2-stimulated osteoblastic differentiation, assessed as induction of alkaline phosphatase activity, in basal, but not pro-osteogenic media. After 10 days, they also inhibited BMP-2-stimulated mineral deposition in pro-osteogenic media. Spp24 had no effect on Erk1/2 phosphorylation, but Spp18 stimulated short-term Erk1/2, MEK 1/2, and p38 phosphorylation. Pertussis toxin and a MEK1/2 inhibitor ablated Spp18-stimulated Erk 1/2 phosphorylation, indicating a role for Gi proteins and MEK1/2 in the Spp18-stimulated Erk1/2 phosphorylation cascade. Truncation products, but not full-length Spp24, stimulated RUNX2, ATF4, and CSF1 transcription. This suggests that Spp24 truncation products have effects on osteoblastic differentiation mediated by kinase pathways that are independent of exogenous BMP/TGF-β cytokines.

Fibroblastic Synoviocytes Secrete Plasma Proteins Via α2‐Macroglobulins Serving as Intracellular and Extracellular Chaperones

“…Through its binding properties, Spp24 modulates the relative availability of TGFβ and BMPs, both of which affect osteoblastic and chondrocytic cells. Spp24 may be proteolytically converted to Spp18, or other size forms, and that directly affect a number of cell types through its cytokine agonist properties and/or signal transduction [Zhao et al, ]. We cannot ascertain at this time whether increased concentrations of Spp24 in SF are ameliorating or detrimental in terms of disease progression, but other investigators have demonstrated that A2M levels increase in post‐traumatic joint fluid and that adding partially purified A2M from normal individuals (which may contain mostly naïve A2M and activated A2M carrying ligands such as Spp24) attenuates the progression of post‐traumatic OA [Wang et al, ].…”

“…For qPCR analysis, Hig‐82 cells were seeded at a density of 10 5 cells/well in 6‐well plates and cultured overnight in F12‐FCS medium at 37°C and 5% CO 2 . Cytokines were added directly to their respective wells to achieve the final working concentrations indicated: 20 pg/ml IL1β, 2 ng/ml IL6, 100 pg/ml TNFα, 10 ng/ml TGFβ1, or 10 ng/ml BMP2 and cultured for an additional 24 h. RNA isolation, cDNA preparation, and qPCR reaction were carried out according to our established protocols [Zhao et al, , ]. Unique oligonucleotide primers for amplification of rabbit plasma protein genes were designed and chemically synthesized by Invitrogen (Supplementary Table SI).…”

Low Mass Blood Peptides Discriminative of Inflammatory Bowel Disease (IBD) Severity: A Quantitative Proteomic Perspective

“…Our data has shown increased levels of kininogen in IBD patients, along with other acute phase proteins such as fibrinogen, ␤-2-microglobulin, ␣-2-macroglobulin, haptoglobin and the apolipoproteins. In particular ␣-2-macroglobulin has previously been implicated as an intra and extracellular chaperone of vesicular traffic in the ER (37,38). All proteins and small peptides have been measured initially from the enriched low mass component of plasma as well as in unfractionated plasma and serum using MRM.…”

Secreted phosphoprotein 24 kD (Spp24) inhibits growth of human osteosarcoma through the BMP-2/Smad signaling pathway

“…Secreted phosphoprotein 24 kD (Spp24) is a bone matrix protein that is likely to have a number of varied functions in the natural bone environment-related not only to its ability to bind several members of the TGFbeta/BMP family of proteins (37,38), but also related to its (or, more precisely, truncated fragments of it) little explored ability to stimulate intracellular signaling pathways (39). We have engineered several protein and peptide products that, while they are thought to function by a single mechanism (BMP binding), operationally have seemingly paradoxically opposite outcomes along a spectrum from the enhancement of BMP activity ("slow-release") to inhibition of BMP activity ("sequestration") (37).…”