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Insulin-like growth factor 1 synergizes with bone morphogenetic protein 7-mediated anabolism in bovine intervertebral disc cells.

ABSTRACT: We undertook this study to assess the therapeutic benefits of intervertebral disc matrix repair and regeneration by evaluating the potential synergistic effect of insulin-like growth factor 1 (IGF-1) and bone morphogenetic protein 7 (BMP-7) on bovine spine discs and by elucidating the relevant molecular/cellular mechanisms.Bovine nucleus pulposus (NP) cells were treated with BMP-7 and IGF-1. The subsequent anabolic effects driven by NP cells were assessed for proteoglycan (PG) synthesis by (35) S-sulfate incorporation and for PG accumulation by dimethylmethylene blue assays. Matrix formation was visualized by particle exclusion assay. Key matrix components and transcription factors were analyzed by real-time reverse transcription-polymerase chain reaction to determine the signaling pathways by which IGF-1 suppresses noggin, a potent inhibitor of BMP-7. Western blotting and nuclear translocation experiments were performed to assess the activation of Smad proteins.Stimulation of bovine NP cells by both IGF-1 and BMP-7 greatly potentiated anabolism through complementary and synergistic mechanisms on matrix formation compared with treatment with either growth factor alone. The exogenously added decoy ligand, noggin, attenuated the anabolic effects of BMP-7, and noggin was substantially increased by BMP-7, suggesting a negative feedback regulatory mechanism. In contrast, IGF-1 significantly suppressed noggin expression via the phosphatidylinositol 3-kinase/Akt pathway and thus potentiated BMP-7 signaling in bovine NP cells. Upon combination treatment, IGF-1 activated Smad2, while BMP-7 activated Smad1/5/8 and Smad3, thus inducing all Smad signaling pathways and mimicking the effects of the combination of transforming growth factor ? and BMP-7Combination growth factor therapy using BMP-7 and IGF-1 may have considerable promise in the treatment of spine disc degeneration.

SUBMITTER: Kim JS

PROVIDER: S-EPMC2995823 | biostudies-literature | 2010 Dec

REPOSITORIES: biostudies-literature

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Insulin-like growth factor 1 synergizes with bone morphogenetic protein 7-mediated anabolism in bovine intervertebral disc cells.

Kim Jae-Sung JS Ellman Michael B MB An Howard S HS van Wijnen Andre J AJ Borgia Jeffrey A JA Im Hee-Jeong HJ

Arthritis and rheumatism 20101201 12

<h4>Objective</h4>We undertook this study to assess the therapeutic benefits of intervertebral disc matrix repair and regeneration by evaluating the potential synergistic effect of insulin-like growth factor 1 (IGF-1) and bone morphogenetic protein 7 (BMP-7) on bovine spine discs and by elucidating the relevant molecular/cellular mechanisms.<h4>Methods</h4>Bovine nucleus pulposus (NP) cells were treated with BMP-7 and IGF-1. The subsequent anabolic effects driven by NP cells were assessed for pr ...[more]

PMID: 20812336

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Project description:Fibroblast growth factor 2 (FGF2) is a growth factor that is immediately released after cartilage injury and plays a pivotal role in cartilage homeostasis. In human adult articular cartilage, FGF2 mediates anti-anabolic and potentially catabolic effects via the suppression of proteoglycan (PG) production along with the upregulation of matrix-degrading enzyme activity. The aim of the present study was to determine the biological effects of FGF2 in spine disc cells and to elucidate the complex biochemical pathways utilized by FGF2 in bovine intervertebral disc (IVD) cells in an attempt to further understand the pathophysiologic processes involved in disc degeneration.We studied the effect of FGF2 on IVD tissue homeostasis by assessing MMP-13 expression (potent matrix-degrading enzyme), PG accumulation, and PG synthesis in the bovine spine IVD, as well as evaluating whether FGF2 counteracts known anabolic factors such as BMP7. To understand the molecular mechanisms by which FGF2 antagonizes BMP7 activity, we also investigated the signaling pathways utilized by FGF2 in bovine disc tissue.The primary receptor expressed in bovine nucleus pulposus cartilage is FGFR1, and this receptor is upregulated in degenerative human IVD tissue compared with normal IVD tissue. Stimulation of bovine nucleus pulposus cells cultured in monolayer with FGF2 augmented the production of MMP-13 at the transcriptional and translational level in a dose-dependent manner. Stimulation of bovine nucleus pulposus cells cultured in alginate beads for 21 days with FGF2 resulted in a dose-dependent decrease in PG accumulation, due at least in part to the inhibition of PG synthesis. Further studies demonstrate that FGF2 (10 ng/ml) antagonizes BMP7-mediated acceleration of PG production in bovine nucleus pulposus cells via the upregulation of noggin, an inhibitor of the transforming growth factor beta/bone morphogenetic protein signaling pathway. Chemical inhibitor studies showed that FGF2 utilizes the mitogen-activated protein kinase and NF-kappaB pathways to upregulate noggin, serving as one potential mechanism for its anti-anabolic effects.FGF2 is anti-anabolic in bovine spine disc cells, revealing the potential of FGF2 antagonists as unique biologic treatments for both prevention and reversal of IVD degeneration.

Project description:Nucleus pulposus (NP) cells have a phenotype similar to articular cartilage (AC) cells. However, the matrix of the NP is clearly different to that of AC suggesting that specific cell phenotypes exist. The aim of this study was to identify novel genes that could be used to distinguish bovine NP cells from AC and annulus fibrosus (AF) cells, and to further determine their expression in normal and degenerate human intervertebral disc (IVD) cells.Microarrays were conducted on bovine AC, AF and NP cells, using Affymetrix Genechip(R) Bovine Genome Arrays. Differential expression levels for a number of genes were confirmed by quantitative real time polymerase chain reaction (qRT-PCR) on bovine, AC, AF and NP cells, as well as separated bovine NP and notochordal (NC) cells. Expression of these novel markers were further tested on normal human AC, AF and NP cells, and degenerate AF and NP cells.Microarray comparisons between NP/AC&AF and NP/AC identified 34 NP-specific and 49 IVD-specific genes respectively that were differentially expressed > or =100 fold. A subset of these were verified by qRT-PCR and shown to be expressed in bovine NC cells. Eleven genes (SNAP25, KRT8, KRT18, KRT19, CDH2, IBSP, VCAN, TNMD, BASP1, FOXF1 & FBLN1) were also differentially expressed in normal human NP cells, although to a lesser degree. Four genes (SNAP25, KRT8, KRT18 and CDH2) were significantly decreased in degenerate human NP cells, while three genes (VCAN, TNMD and BASP1) were significantly increased in degenerate human AF cells. The IVD negative marker FBLN1 was significantly increased in both degenerate human NP and AF cells.This study has identified a number of novel genes that characterise the bovine and human NP and IVD transcriptional profiles, and allows for discrimination between AC, AF and NP cells. Furthermore, the similarity in expression profiles of the separated NP and NC cell populations suggests that these two cell types may be derived from a common lineage. Although interspecies variation, together with changes with IVD degeneration were noted, use of this gene expression signature will benefit tissue engineering studies where defining the NP phenotype is paramount.

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Project description:Bovine caudal discs have been widely used in spine research due to their increased availability, large size, and mechanical and biochemical properties that are comparable to healthy human discs. However, despite their extensive use, the radial variations in bovine disc composition have not yet been rigorously quantified with high spatial resolution. Previous studies were limited to qualitative analyses or provided limited spatial resolution in biochemical properties. Thus, the main objective of this study was to provide quantitative measurements of biochemical composition with higher spatial resolution than previous studies that employed traditional biochemical techniques. Specifically, traditional biochemical analyses were used to measure water, sulfated glycosaminoglycan, collagen, and DNA contents. Gravimetric water content was compared to data obtained through Raman spectroscopy and differential scanning calorimetry. Additionally, spatial distribution of lipids in the disc's collagen network was visualized and quantified, for the first time, using multi-modal second harmonic generation (SHG) and Coherent anti-Stokes Raman (CARS) microscopy. Some heterogeneity was observed in the nucleus pulposus, where the water content and water-to-protein ratio of the inner nucleus were greater than the outer nucleus. In contrast, the bovine annulus fibrosus exhibited a more heterogeneous distribution of biochemical properties. Comparable results between orthohydroxyproline assay and SHG imaging highlight the potential benefit of using SHG microscopy as a less destructive method for measuring collagen content, particularly when relative changes are of interest. CARS images showed that lipid deposits were distributed equally throughout the disc and appeared either as individual droplets or as clusters of small droplets. In conclusion, this study provided a more comprehensive assessment of spatial variations in biochemical composition of the bovine caudal disc.

Dataset Information

Insulin-like growth factor 1 synergizes with bone morphogenetic protein 7-mediated anabolism in bovine intervertebral disc cells.

Publications

Insulin-like growth factor 1 synergizes with bone morphogenetic protein 7-mediated anabolism in bovine intervertebral disc cells.

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