Phospholipase A₂ activating protein (PLAA) is required for 1α,25(OH)₂D₃ signaling in growth plate chondrocytes

Abstract

Phospholipase A2 (PLA2) is pivotal in the rapid membrane-mediated actions of 1,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3]. Microarray analysis indicated that PLA2 activating protein (PLAA) mRNA is upregulated 6-fold before rat growth plate cells exhibit 1alpha,25(OH)2D3-dependent protein kinase C (PKC) increases, suggesting that it plays an important role in 1alpha,25(OH)2D3's mechanism of action. PLAA mRNA was confirmed in 1alpha,25(OH)2D3-responsive growth zone (prehypertrophic and upper hypertrophic cell zones) chondrocytes by RT-PCR and Northern blot in vitro and by in situ hybridization in vivo. PLAA protein was shown by Western blot and immunohistochemistry. PLAAs role in 1alpha,25(OH)2D3 signaling was evaluated in growth zone cell cultures using PLAA peptide. Arachidonic acid release was increased as was PLA2-specific activity in plasma membranes and matrix vesicles. PKCalpha, but not PKCbeta, PKCepsilon, or PKCzeta, was increased. PLAAs effect was comparable to that of 1alpha,25(OH)2D3 and was additive with 1alpha,25(OH)2D3. PLA2 inhibitors quinacrine and AACOCF3, and cyclooxygenase inhibitor indomethacin blocked the effect of PLAA peptide on PKC, indicating arachidonic acid and its metabolites were involved. This was confirmed using exogenous arachidonic acid. Prostaglandin acted via EP1 based on inhibition by SC19220 and not via EP2 since AH6809 had no effect. Like 1alpha,25(OH)2D3, PLAA peptide also increased activity of phospholipase C-specific activity via beta-1 and beta-3 isoforms, but not delta-1 or gamma-1; the effect of PLAA was via lysophospholipid but not via arachidonic acid. PLAA peptide decreased [3H]-thymidine incorporation to 50% of the decrease caused by 1alpha,25(OH)2D3. In contrast, PLAA peptide increased alkaline phosphatase-specific activity and proteoglycan production in a manner similar to 1alpha,25(OH)2D3. This indicates that PLAA is a specific activator of PLA2 in growth plate chondrocytes, and suggests that it mediates the membrane effect of 1alpha,25(OH)2D3, thereby modulating physiological response.

Protein-disulfide Isomerase-associated 3 (Pdia3) Mediates the Membrane Response to 1,25-Dihydroxyvitamin D3 in Osteoblasts

“…Importantly, the results demonstrate that Pdia3-dependent signaling results in changes in gene expression, via phosphorylation of transcription factors such as ERK1/2. Taking these observations together with our previous observations using growth plate chondrocytes (16,27,28) and osteoblasts (19,21), we propose a mechanism initiated at the plasma membrane and culminating in downstream genomic regulation (Fig. 7).…”

“…This results in arachidonic acid release; the arachidonic acid is processed further to PGE 2 . In growth plate chondrocytes (1,16,18). PGE 2 binds its G-protein coupled receptor and activates PLC.…”

ESWT Enhances Expression of Pdia-3 which is a Key Factor of 1α,25-Dihydroxyvitamin D3 Rapid Membrane Signaling Pathway in Treatment of Early Osteoarthritis Knee

“…Our current study provides the first evidence from immunohistochemistry that shockwave therapy can induce articular cartilages expression of Pdia-3, the critical transcription factor responsible for the matrix formation of chondrocyte. Recent studies reported that 1α,25(OH)2D3 rapidly stimulated membrane signaling via Pdia-3 dependent activation in growth zone chondrocytes and promotes the production of matrix protein [11,13,20,22,42,43]. The present study showed the decrease of cartilage matrix loss and increased aggrecan and collagen II expression in shockwave group.…”

1,25-Dihydroxy Vitamin D₃ Is an Autocrine Regulator of Extracellular Matrix Turnover and Growth Factor Release via ERp60-Activated Matrix Vesicle Matrix Metalloproteinases

“…In contrast to its effects on PKC isoforms, 1 ␣ ,25(OH) 2 D 3 stimulates PLA 2 activity in both membrane compartments [Schwartz et al, 2005], suggesting an alternate hypothesis. In this hypothesis, activation of PLA 2 in the PM is rapidly downregulated, but in the MVs this does not occur and the resulting production of lysophospholipids causes a loss of membrane integrity and eventual release of MMPs into the ECM.…”