Immunolocalization and Biochemical Evidence of Pregnancy-Associated Plasma Protein-A in the Intervertebral Disc

Study Design. Studies were approved by the authors’ Human Subjects Institutional Review Board and Institutional Animal Care and Use Committee. Anulus tissue was used in studies of the immunocytochemical localization of pregnancy-associated plasma protein A (PAPP-A) in disc tissue from the sand rat and from human disc surgical specimens and specimens from control donors. Cultured human disc cells were also tested for production of PAPP-A. Objectives. (1) To determine the immunohistochemical localization of PAPP-A in human and sand rat discs; (2) To test for gene expression of PAPP-A in the human disc in vivo and in vitro production by cultured cells; and (3) To test for expression of insulin-like growth factor binding proteins (IGFBP)-2, -4, and -5 in vivo and in vitro by human disc cells. Summary of Background Data. PAPP-A is a metalloproteinase expressed by several cell types, including fibroblasts, osteoblasts, and smooth muscle cells. PAPP-A has an extremely important role because it cleaves IGFBP-2, -4, and -5 in the extracellular matrix, thereby increasing the bioavailability of IGF to nearby cells. Methods. Specimens of human disc tissue and lumbar discs from sand rats were assessed for immunocytochemical localization of PAPP-A, and the percentage of positive cells determined. Human disc cells in three-dimensional culture were assessed for production of PAPP-A using an enzyme linked immunosorbent assay. Molecular gene expression studies were carried out using microarray analysis. Results. Positive cytoplasmic immunolocalization of PAPP-A was present in the majority of cells of the human and sand rat outer anulus (OA). In the human outer anulus, the percentage of cells positive for PAPP-A localization did not differ in Grades I–II discs vs. Grades III–V discs (OA: 77.4% ± 10.5 vs. 75.1% ± 7.4 [mean ± SEM] respectively). In the inner anulus, however, the percentage of cells positive for PAPP-A localization in more degenerate discs was significantly greater than the percentage in healthier discs (60.7% ± 10.1 vs. 15.6 ± 5.4, P = 0.024). % positive cells in the inner anulus correlated significantly with disc grade (r2 = 0.579; P = 0.01). Over a 5-day three-dimensional culture period, human anulus cells produced and secreted abundant PAPP-A into the culture media. Molecular studies confirmed the expression of IGFBP-2, -4, and -5 both in vivo and in vitro. Conclusions. Data provide important new insights into disc cell expression of PAPP-A at the translational level. The presence of a significantly greater proportion of cells positive for PAPP-A in the inner anulus of more degenerate Grade III–V discs compared with healthier Grade I–II discs supports our previous observation of increased gene expression of PAPP-A in more degenerated discs. Biochemical data shown here documented production of PAPP-A by disc cells in vitro. Production of PAPP-A by disc cells is important since PAPP-A cleaves IGF-binding proteins, and makes IGF-I, a potent mitogen and antiapoptotic agent, available to cells. Future studies are indicated to further investigate PAPP-A and IGF-BP function in the disc.