Abstract
Introduction
Immature nucleus pulposus (NP) cells of the intervertebral disc (IVD) are large, vacuolated cells that form cell clusters with strong cell-cell interactions.1 With maturation and aging, NP cells lose their ability to form these cell clusters, with aging-associated changes in NP cell phenotype, morphology, and proteoglycan synthesis that may contribute to IVD degeneration.2-4 Therefore it is important to understand the mechanisms governing immature NP cell cluster behavior toward the goal of revealing factors that can promote immature, healthy NP cell phenotypes.
Prior work has shown healthy, immature NP cells preferentially bind laminin proteins, exhibit potential for cell cluster formation, and maintain matrix production when cultured upon soft, laminin-containing substrates.2 N-cadherin has been identified as a cell-cell adhesion marker that is present in immature NP cells, but disappears with age.5-6 The goal of this study was to determine if N-cadherin was an important molecule in regulating cell-cell interactions in immature NP cell cluster formation and to test for a regulatory role in maintaining an immature NP phenotype for IVD cells in vitro.
Materials and Methods
Porcine nucleus pulposus (NP) cells were isolated from IVD of immature pigs (3-6 mos).2 Laminin-rich substrates were constructed from basement membrane extract (BME, 13.7 mg/mL, Trevigen) to generate soft (300 Pa) or stiff (BME-coated glass, > 2,900 Pa) substrates; similar substrates were constructed of “soft” and “stiff” type I collagen (col 1, 4 mg/mL, Sigma). NP cells were plated (65,000 cells/cm2) on the substrates with the following treatment conditions for up to 4 days: N-cadherin (40 μg/mL) blocking antibody, E-cadherin (20 μg/mL) blocking antibody or no treatment control. NP cells were stained for polymerized F-actin (phalloidin, Invitrogen), anti-N-cadherin (Abcam), or anti-E-cadherin (Abcam) antibodies with propidium iodide nuclei counterstain. Changes in matrix production were analyzed in collected media and recovered cells via biochemical assays for sGAG (DMMB assay) and DNA (picogreen). Additionally, gene expression for immature NP markers, N-cadherin, Brachyury-T, aggrecan and type I collagen, were analyzed via quantitative real-time PCR.
Results
NP cells cultured upon soft BME substrates maintained their rounded morphology at all times with formation of 3D cell clusters (Fig. 1A). On stiff BME and all col 1 substrates, NP cells spread out and attached with formation of numerous actin stress fibers. Higher N-cadherin expression was observed on soft BME compared with all other substrates; E-cadherin expression was absent in porcine NP cells on all substrates. Additionally, significantly higher matrix production was observed on soft BME substrates compared with soft col 1 (Fig. 1B), and higher gene expression for NP markers was observed on soft BME substrates compared with all other substrates (Fig. 1C). NP cell cluster formation, proteoglycan synthesis and NP marker gene expression on soft BME substrates was influenced by N-cadherin blocking antibody but not E-cadherin blocking antibody (Fig. 1A-C).
Changes in NP cell morphology and phenotype with cadherin blocking antibodies. (A) Phalloidin immunostaining (green) for porcine NP cells on soft and stiff BME substrates (red = nuclei, scale bar = 50 μm). (B) Proteoglycan production (sGAG/DNA) (*p<0.05, 2-way ANOVA with Tukey's posthoc analysis). (C) Gene expression of NP markers
Conclusion
Soft BME substrates promote NP cell clustering in vitro with an associated expression of N-cadherin. In NP cells, it appears that N-cadherin-mediated signaling helps promote matrix production and elevated expression for multiple molecular markers of an immature NP phenotype. Treatment with an N-cadherin blocking antibody resulted in loss of all features of the immature NP cell on soft BME substrates. These findings establish the importance of N-cadherin in mediating immature NP cell cluster formation and maintenance of the immature phenotype. Ongoing studies aim to understand the downstream signaling mechanisms governed by N-cadherin-mediated signaling that promote the immature NP phenotype.
None declared
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