Expression of Laminin Isoforms, Receptors, and Binding Proteins Unique to Nucleus Pulposus Cells of Immature Intervertebral Disc

Abstract

Intervertebral disc (IVD) disorders are believed to be related to aging-related cell loss and phenotypic changes, as well as biochemical and structural changes in the extracellular matrix of the nucleus pulposus (NP) region. Previously, we found that the laminin γ1 chain was more highly expressed in immature NP porcine tissues, in parallel with the expression pattern for a laminin receptor, integrin α6 subunit, as compared to adjacent anulus fibrosus region; suggesting that cell-matrix interactions may be unique to the immature NP. However, the identity of laminin isoforms specific to immature or mature NP tissues, their associated receptors and functional significance are still poorly understood. In this study, we evaluated the zonal-specific expression of the laminin chains, receptors (i.e. integrins) and other binding proteins in immature tissue and isolated cells of rat, porcine and human intervertebral disc, towards the goal of revealing features of cellular environment and cell-matrix interactions in the immature NP. Results from both immuno-histochemical staining and flow cytometry analysis found that NP cells expressed higher levels of the laminin α5 chain, laminin receptors (integrin α3, α6, β4 subunit and CD239) and related binding proteins (CD151), as compared to cells from adjacent anulus fibrosus. These differences suggest that laminin interactions with NP cells are distinct from that of the anulus fibrosus, and that laminins may be important contributors to region-specific IVD biology. The revealed laminin isoforms, their receptors and related binding proteins may be used as distinguishing features of these immature NP cells in the intervertebral disc.

The Role Of Extracellular Matrix Elasticity and Composition In Regulating the Nucleus Pulposus Cell Phenotype in the Intervertebral Disc: A Narrative Review

“…In the young, healthy human, the NP ECM elasticity ranges from 0.3-5 kPa [56,57] and is comprised primarily of water (70-90% of wet weight), proteoglycans (65% of dry weight), and randomly oriented type II collagen (15-20% dry weight) [58][59][60]. Other compositionally minor (though potentially critical functionally) ECM components of the NP include elastin, small proteoglycans, and minor coUagens (types III, VI, IX) [61][62][63], and laminins [25,64,65]. Many of these ECM constituents can directly interact with NP cells through cell-surface receptors, if resident within the cell pericellular matrix or multicell cluster (e.g., type VI collagen, N-cadherin [53]); altemately, these ECM constituents can exert a mechanical influence upon NP cells through regulating nutrient transport, hydration (e.g., fixed charge density) and swelling pressure [66].…”

An understanding of intervertebral disc development, maturation and cell phenotype provides clues to direct cell-based tissue regeneration therapies for disc degeneration

“…This, in turn, will allow a more thorough understanding, at the molecular level, of the fate and role of notochordal or notochord-derived cells in the IVD and provide further insight into ways to tackle disc degeneration. Lamins are heterodimeric proteins involved in cell adhesion, migration, proliferation, differentiation and survival [74,77] Pending questions and potential clinical implications…”

“…Integrins and CD239 are expressed by NP cells and are responsible for their adhesion to laminins [74,77] Integrin subunits a3, a6 and ß4)…”

“…For this to be possible, a comprehensive phenotypic characterization of notochordal cells, and especially of markers specific to the notochordal cells in the human IVD would be required. To date, several attempts have been made to identify notochordal markers, either by comparing the gene expression of immature notochordal rat NP with its costal cartilage [73], rat NP tissue with different degrees of maturity [74][75][76], comparing immature pig NP with AF cells [74,77], sorted large and granular with smaller, less granular pig NP cells [78], immature human NP with AF cells [74] or NP from human juvenile scoliotic and adult discs [79] (Tables 1, 2, 3, 4 list putative notochordal/ immature NP markers identified in different species and describe their relevance to the IVD field). Proposed rat notochordal markers identified in these studies were CD55 [73], brachyury, neuropilin (Nrp-1), CD221, BASP-1 [76], N-Cad [73,76], TGF-b, BMP-6 and CTGF [75].…”

Reconstruction of an In Vitro Niche for the Transition from Intervertebral Disc Development to Nucleus Pulposus Regeneration

“…Currently, at least 15 isoforms of laminin have been identified; these are expressed differently in various tissues [83,84]. In NPCs, a higher expression of the laminin a5 chain, laminin receptor integrin subunits a3, a6, and b4 along with CD239 and CD151 were found compared to cells from the AF [85]. Gilchrist et al also examined the cell-matrix interactions and found a faster cell spreading and a higher resistance to detachment on laminin isoforms LM-511 and LM-332, in contrast to LM-111, fibronectin, and collagen II [86].…”