Glutamate inhibits chondral mineralization through apoptotic cell death mediated by retrograde operation of the cystine/glutamate antiporter.

Although we have previously demonstrated the functional significance of excitatory amino acid transporters as well as glutamate (Glu) receptors (GluRs) expressed by chondrocytes, little attention has been paid to the possible expression of the cystine/Glu antiporter responsible for the bi-directional transmembrane transport of Glu in chondrocytes to date. In organotypic cultured mouse embryonic metatarsals isolated before vascularization, the chondral mineralization was significantly decreased in the presence of Glu at a high concentration. Apoptotic cells were detected within the late proliferating and prehypertrophic chondrocytic layers in metatarsals cultured in the presence of Glu.

Abolition of chondral mineralization by group III metabotropic glutamate receptors expressed in rodent cartilage.

1 Previous studies have demonstrated the functional expression by osteoblasts of glutamate (Glu) signaling machineries responsible for the stimulation of cell proliferation and differentiation in bone, while there is no information available on the expression of the Glu signaling system by cartilage to date. 2 In cultured mouse embryonic metatarsals isolated before vascularization, chondral mineralization was almost completely inhibited in the presence of the group III metabotropic Glu receptor (mGluR) agonist L-(1)-2-amino-4-phosphonobutyrate (L-AP4) in a manner sensitive to an antagonist, with the total length being unchanged. 3 A group II mGluR agonist was similarly more effective in inhibiting the mineralization than a group I mGluR agonist, while none of ionotropic GluR agonists drastically affected the mineralization.

Release of endogenous glutamate by AMPA receptors expressed in cultured rat costal chondrocytes.

We have previously demonstrated the release of endogenous glutamate by activation of DL-alpha-amino-3-hydroxy-5-methylisoxasole-4-propionate (AMPA) receptors expressed by bone, while there is no information available on the possible functional expression of glutamatergic signaling molecules in cartilage to date. In rat costal chondrocytes cultured for 4 to 28 d, expression of mRNA was seen for several chondral marker genes including sox9, runt-related gene 2/core binding factor alpha-1 (Runx-2/Cbfa-1), type II collagen and aggrecan, but not for the adipocyte marker gene peroxisome proliferator-activated receptor gamma (PPARgamma). Expression of mRNA was drastically increased for Runx-2/Cbfa-1 during culturing from 7 to 14 d with a gradual increase thereafter up to 28 d, while a transient increase was seen in mRNA expression for both type-II collagen and sox-9 on 14 d and for aggrecan on 7 d respectively, in chondrocytes cultured for a period up to 28 d.

Functional expression of particular isoforms of excitatory amino acid transporters by rodent cartilage.

In the present study, we have attempted to demonstrate functional expression by the rodent cartilage of particular isoforms of excitatory amino acid transporters (EAATs) essentially required for central glutamatergic signal termination. Constitutive expression of mRNA was shown for the first time with the neuronal EAAT subtype excitatory amino acid carrier-1 (EAAC1), in addition to glial subtypes such as glutamate aspartate transporter (GLAST) and glutamate transporter-1 (GLT-1), in rat costal chondrocytes cultured for 7-21 days on reverse transcription polymerase chain reaction (RT-PCR). Western blotting analysis confirmed the expression of corresponding proteins for both GLAST and GLT-1 in cultured chondrocytes.

Demonstration of expression of mRNA for particular AMPA and kainate receptor subunits in immature and mature cultured rat calvarial osteoblasts.

Reverse transcription polymerase chain reaction revealed expression of mRNA for particular subunits of ionotropic glutamate receptors (iGluR) in primary cultures of rat calvarial osteoblastic cells under immature to mature states. These included GluR3, KA1 and KA2 subunits, in addition to NR1 and NR2D subunits. These results suggest that glutamate may play an unidentified role in mechanisms associated with cellular development through particular subunits of iGluR in rat calvarial osteoblasts.

Cell death by pyruvate deficiency in proliferative cultured calvarial osteoblasts.

Cell survival was significantly decreased in primary cultured rat calvarial osteoblasts in vitro at Day 0, 1, and 3 by replacement of the standard culture medium (alpha-modified minimum essential medium; alpha-MEM) with Dulbecco's modified eagle's medium (DMEM). Decreased cell survival was also observed following medium replacement in cultures of murine calvaria-derived osteoblastic cell line MC3T3-E1. Staining with Hoechst33342 revealed apoptotic cells with fragmented or condensed nuclei, while a fraction of the cell culture was stained with propidum iodide, indicating necrosis.