Effect of Parathyroid Hormone on MAPK Signaling and Calcification Markers in Human Intervertebral Disk Cells

Introduction Back pain is strongly associated with degeneration of the intervertebral disk (IVD), which is associated with ongoing mineral deposition.1 The presence of calcium deposits and type X collagen (COL X) and the level of the indicators of calcification potential (alkaline phosphatase (ALP), Ca2 + ions and Pi) were consistently higher in degenerative and scoliotic discs. We also showed that in mesenchymal stem cells (MSCs), parathyroid hormone (PTH 1–34) inhibits the expression of COL X while promoting type II collagen (COL II) expression, thereby preventing endochondral ossification.2 In this study, we investigated the effect of PTH on expression of COLII, COLX, and ALP in human IVD cells and analyzed the potential mechanisms related to its effect. Materials and Methods Human lumbar IVDs from a donor without spinal pathology were obtained within 24 hours after death. Nucleus pulposus (NP) and annulus fibrosus (AF) tissues from the IVDs were digested and the corresponding NP and AF primary cells were isolated as previously described.3These cells were cultured in complete DMEM to 90% confluence. Then the cells were incubated overnight in serum-free medium followed by treatment with 100 nM PTH 30 minutes to 48 hours. Protein expression was analyzed by immunoblotting using specific antibodies to COL I and COL II (Abcam, Cambridge, MA), COL X, and PTH receptor 1 (Sigma Aldrich). Expression and phosphorylation of AKT and MAPKs was assessed by using specific corresponding antibodies (Cell Signaling, Danvers, MA). Alkaline phosphatase activity was measured colorimetrically using the StemTAG kit (Cell Biolabs, San Diego, CA, USA) and Ca2 + release from cells was measured using calcium assay kit (Cayman Chemical, Ann Arbor, MI, USA). Statistical analyses were done using one-way ANOVA, posthoc tests p