Impaired osteoblast differentiation in annexin A2- and -A5-deficient cells.

Damian C Genetos,Norman J Karin,Clare E Yellowley,Alice Wong,Thomas J Weber,Carlos M Isales

doi:10.1371/journal.pone.0107482

Damian C Genetos, Norman J Karin + Show 4 more

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https://doi.org/10.1371/journal.pone.0107482

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Abstract

Annexins are a class of calcium-binding proteins with diverse functions in the regulation of lipid rafts, inflammation, fibrinolysis, transcriptional programming and ion transport. Within bone, they are well-characterized as components of mineralizing matrix vesicles, although little else is known as to their function during osteogenesis. We employed shRNA to generate annexin A2 (AnxA2)- or annexin A5 (AnxA5)-knockdown pre-osteoblasts, and determined whether proliferation or osteogenic differentiation was altered in knockdown cells, compared to pSiren (Si) controls. We report that DNA content, a marker of proliferation, was significantly reduced in both AnxA2 and AnxA5 knockdown cells. Alkaline phosphatase expression and activity were also suppressed in AnxA2- or AnxA5-knockdown after 14 days of culture. The pattern of osteogenic gene expression was altered in knockdown cells, with Col1a1 expressed more rapidly in knock-down cells, compared to pSiren. In contrast, Runx2, Ibsp, and Bglap all revealed decreased expression after 14 days of culture. In both AnxA2- and AnxA5-knockdown, interleukin-induced STAT6 signaling was markedly attenuated compared to pSiren controls. These data suggest that AnxA2 and AnxA5 can influence bone formation via regulation of osteoprogenitor proliferation, differentiation, and responsiveness to cytokines in addition to their well-studied function in matrix vesicles.

Highlights

Annexins comprise a class of calcium-dependent, phospholipidbinding proteins that are broadly expressed in eukaryotic cells
There was a significant reduction (.80%) in annexin A2 (AnxA2) mRNA expression in AnxA2 knockdown cells (AnxA2kd) cells compared to stablytransfected with pSIREN vector (Si)-transfected controls (Figure 1A), and there was no compensatory change in annexin A5 (AnxA5) mRNA in AnxA2kd cells
Both AnxA2 and AnxA5 are present in matrix vesicles isolated from both chondrocytes and osteoblasts, where they are thought to act as membrane channels to allow Ca2+ influx and hydroxyapatite crystal formation

Summary

Introduction

Annexins comprise a class of calcium-dependent, phospholipidbinding proteins that are broadly expressed in eukaryotic cells. They are predominately localized within the cell, where they mediate such cellular processes as exocytosis and endocytosis, membrane structure and generation of lipid rafts, formation or regulation of ion channels, and cytokinesis. Of the 12 Annexins expressed in mammals, Annexins A1, A2, A4, A5, A6 and A7 are expressed within cells of the chondrogenic and osteoblastic lineage [5,6,7] To date, their function within these cells has primarily focused upon a putative role in matrix mineralization. We have reported that AnxA5 is involved in transducing a biophysical signal–fluid shear stress–

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