N-methyl-D-aspartate (NMDA) receptor expression and function is required for early chondrogenesis

Abstract

BackgroundIn vitro chondrogenesis depends on the concerted action of numerous signalling pathways, many of which are sensitive to the changes of intracellular Ca2+ concentration. N-methyl-D-aspartate (NMDA) glutamate receptor is a cation channel with high permeability for Ca2+. Whilst there is now accumulating evidence for the expression and function of NMDA receptors in non-neural tissues including mature cartilage and bone, the contribution of glutamate signalling to the regulation of chondrogenesis is yet to be elucidated.MethodsWe studied the role of glutamatergic signalling during the course of in vitro chondrogenesis in high density chondrifying cell cultures using single cell fluorescent calcium imaging, patch clamp, transient gene silencing, and western blotting.ResultsHere we show that key components of the glutamatergic signalling pathways are functional during in vitro chondrogenesis in a primary chicken chondrogenic model system. We also present the full glutamate receptor subunit mRNA and protein expression profile of these cultures. This is the first study to report that NMDA-mediated signalling may act as a key factor in embryonic limb bud-derived chondrogenic cultures as it evokes intracellular Ca2+ transients, which are abolished by the GluN2B subunit-specific inhibitor ifenprodil. The function of NMDARs is essential for chondrogenesis as their functional knock-down using either ifenprodil or GRIN1 siRNA temporarily blocks the differentiation of chondroprogenitor cells. Cartilage formation was fully restored with the re-expression of the GluN1 protein.ConclusionsWe propose a key role for NMDARs during the transition of chondroprogenitor cells to cartilage matrix-producing chondroblasts.

The temporal transcriptomic signature of cartilage formation

“…Photomicrographs of the native micromass cultures were taken using an inverted microscope, and after staining with haematoxylin and eosin as described previously (20). Hyaline cartilage-specific ECM production was qualitatively analysed as follows: dimethyl methylene blue dye (DMMB; pH 1.8; Sigma-Aldrich) was applied to cultures from day 0 to day 6 as previously described (19). Photomicrographs of the stained specimens were obtained using an Olympus BX53 camera on a Nikon Eclipse E800 microscope (Nikon Corporation, Tokyo, Japan).…”

“…LMP cells in HDCs first proliferate and then differentiate into matrix-producing chondroblasts during the first 3 days of culture, and a well-detectable hyaline cartilage-specific ECM was formed on the 6 th day of culture. HDCs were established as previously described (15,18,19). Briefly, chondroprogenitor cells were isolated from the developing limb buds of chicken embryos at Hamburger-Hamilton stages 22-24. The in vitro work on early-stage (4.5-day-old) chicken embryos does not require approval from the Ethics Committee of the University of Debrecen.…”

Ca2+-Activated K+ Channels in Progenitor Cells of Musculoskeletal Tissues: A Narrative Review

“…NMDA receptors expressed by mature healthy and OA chondrocytes are likely involved in mechanotransduction pathways [ 33 , 34 ] and/or in maintaining tissue homeostasis through regulating the chondrocyte circadian clock [ 35 , 36 ]. NMDAR expression and function are also required for chondrogenic differentiation [ 37 ].…”

Analysis of Gene Expression Patterns of Epigenetic Enzymes Dnmt3a, Tet1 and Ogt in Murine Chondrogenic Models

“…On the designated days of culturing, both primary and cell line-based micromass cultures were washed with physiological NaCl two times and stored at −80 • C. Total RNA from micromass cultures established from C3H10T1/2 BMP-2 cells and primary chondrifying micromass cultures was isolated as previously described [32]. Briefly, cultures were mixed with TRI Reagent (Applied Biosystems, Foster City, CA, USA).…”