Phosphoinositide 3-kinase/Akt signaling is essential for prostaglandin E2-induced osteogenic differentiation of rat tendon stem cells

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Abstract

Tissue calcification is a typical histopathological feature of tendinopathy. The osteogenic differentiation of tendon stem cells (TSCs) induced by inflammatory mediators is believed to play a key role in this process. Previous studies showed that the major inflammatory mediator prostaglandin E2 (PGE2) induced osteogenic differentiation of TSCs via bone morphogenetic protein (BMP)-2 production. Using a rat TSC culture model, we showed that PGE2 induced BMP-2 production through up-regulation of BMP-2 mRNA expression. PGE2 activated Akt, but not extracellular-signal-regulated kinase, in TSCs. Increased BMP-2 mRNA expression mediated by PGE2 was prevented by phosphoinositide 3-kinase (PI3K) and Akt inhibitors, but not by a MEK inhibitor. Furthermore, in the presence of exogenous BMP-2, PI3K and Akt inhibitors blocked Runx2 and osteocalcin expression, although BMP-2 did not activate Akt. BMP-2-induced alkaline phosphatase activity and mineralization were also inhibited by PI3K and Akt inhibitors. However, these inhibitors did not block activation of Smad, implying that Akt was involved downstream of Smad. Taken together, these results indicate that the PI3K-Akt signaling cascade is essential for PGE2-induced BMP-2 production and BMP-2-mediated osteogenic differentiation, suggesting that PI3-kinase-Akt signaling contributes to the formation of calcified tissues in tendinopathy.

Highlights

► PI3-kinase/Akt signaling not ERK signaling, is involved in PGE2-induced osteogenic differentiation of rat tendon stem cells. ► Akt activation is involved in PGE2-induced BMP-2 production. ► PI3-kinase/Akt signaling is essential in BMP-2-induced osteogenic differentiation. ► PI3-kinase or Akt inhibitor does not inhibit BMP-2-activated Smad phosphorylation.

Introduction

Tendons are fibrous bands of connective tissue that connect muscles to bones and transmit muscular forces to the bones, allowing joint motion and subsequent body movement. However, tendons are constantly subjected to large mechanical loading and are thus prone to pathological changes, known as tendinopathy [1]. Tendinopathy is a collective term for tendon disorders involving inflammation and/or degeneration.

Although the precise pathogenic mechanisms of tendinopathy remain unclear, the typical histopathological features include accumulation of lipid cells, mucoid degeneration, tissue calcification, or some combination of these, suggesting that tendons contain cells with multi-differentiation potentials. Tendon stem cells (TSCs) have been identified in humans [2], mice [3], rabbits [4] and rats [5]. These stem cells can differentiate into non-tenocyte lineages such as adipocytes, chondrocytes, and osteocytes under intensive, repetitive mechanical loading [2], [6].

Previous studies suggested that prostaglandin E2 (PGE2), a major inflammatory mediator of pain and acute inflammation in injured tendons [7], [8], [9], was markedly increased in tendons subjected to repetitive mechanical loading conditions in vivo [6]. PGE2 treatment may result in degenerative changes of the tendon, partly by inducing differentiation of TSCs into adipocytes and osteocytes [6]. Moreover, PGE2 treatment of human TSCs (hTSCs) induced the production of bone morphogenetic protein-2 (BMP-2) in culture, and BMP-2 may mediate PGE2-induced osteogenic differentiation of hTSCs [10].

BMPs are a group of polypeptides within the transforming growth factor (TGF)-superfamily, originally named for their ability to promote ectopic bone formation [11]. Among the many BMPs, BMP-2 causes mesenchymal stem cells, and even myogenic cells, to differentiate into osteoblasts [12], [13]. Like other members of the TGF-β superfamily, BMPs exert their effects via type I and type II transmembrane serine/threonine kinase receptors [14], [15], and activate the intracellular signaling molecules Smad1, Smad5 and Smad8 through their serine phosphorylation [16]. Activated Smads induce the expression of Runx2, the master regulator of osteogenesis [17]. Smad and Runx2 then collaboratively induce other genes in differentiating osteoblasts [18]. However, the molecular mechanisms through which PGE2 induces the production of BMP-2 and the subsequent osteogenic differentiation of TSCs remains unknown. PGE2 exerts its downstream effects by signaling through four distinct G-protein-coupled E-prostanoid receptors (EP1–EP4) [19] and then initiates a series of signaling pathways. Although several signaling pathways mediate osteogenic differentiation, a growing literature supports critical roles for the mitogen-activated protein kinase (MAPK) pathway (MEK-ERK) [20], [21], [22] and phosphoinositide 3-kinase (PI3K)-Akt signaling [23], [24].

We investigated the involvement of these pathways in PGE2-induced osteogenic differentiation using cultured rat TSCs, and defined a key role for the PI3-kinase-Akt pathway in PGE2-induced BMP-2 production and BMP-2-mediated osteogenic differentiation.

Section snippets

Isolation and culture of rat TSCs

Three 8-week-old Sprague–Dawley rats weighing 250–300 g were used. All experiments were approved by the Animal Research Ethics Committee, Third Military Medical University, China. The isolation and culture of rat TSCs were performed as described previously [5]. Briefly, the whole piece of intact flexor tendon was excised from both limbs of each rat following euthanasia. Only the midsubstance tissue was collected and peritendinous connective tissue was removed carefully. The tissues were minced

BMP-2 mediates PGE2-induced osteogenic differentiation

Rat TSCs exhibited slender fibroblast-like shapes with an irregular distribution (Fig. 1A). The stem cell markers Oct-4, SSEA-4, and nucleostemin were expressed in TSCs, as detected by immunofluorescence (Fig. 1B–D).

After culture of TSCs in PGE2-containing growth medium for 14 days, mineralization of the extracellular matrix and bone-specific alkaline phosphatase activity were significantly increased (Fig. 1E and F), compared to the control group without PGE2 treatment. We investigated the

Discussion

Chronic mechanical loading of tendons is considered to be the major cause of tendon inflammation and degeneration [27], which are involved in the development of tendinopathy [6], [28]. Tissue calcification is a typical histopathological feature of tendinopathy, and the osteogenic differentiation of TSCs induced by inflammatory mediators may play a key role in this process [6], [10]. As a major inflammatory mediator, PGE2 is present in tendons subjected to repetitive mechanical loading

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    This work was supported in part by the National Nature Science Foundation of China (grant nos. 30872620 and 81071464), the Nature Science Foundation of Chongqing (grant no. CSTC2011BA5010) and the National Eleventh Five-year Plan (grant no. 06MB245).

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