Inhibition of p38 MAPK signaling in chondrocyte cultures results in enhanced osteogenic differentiation of perichondral cells

Abstract

Chondrocytes and osteoblasts originate from the same progenitor cell; however, both are characterized by distinct gene expression profiles once they are differentiated. Signals from differentiating chondrocytes, such as Indian hedgehog (Ihh), regulate the differentiation of osteoblast precursor cells. The MAPK pathways play important roles in controlling the differentiation of both chondrocytes and osteoblasts, with the p38 pathway being particularly relevant in skeletal cells. In the present study, we investigated the effects of p38 inhibition on osteoblastic marker gene expression in chondrocyte cultures. Using high-density micromass cultures of mesenchymal cells as well as chondrocytes that had differentiated in vivo and were maintained in short-term monolayer culture, we demonstrate elevated Runx2, Osterix and Osteocalcin transcript levels in chondrocyte cultures upon inhibition of p38 activity with the pharmacological inhibitor PD169316. Osteocalcin immunolocalization was restricted to perichondral/periosteal cells in micromass cultures, suggesting that inhibition of p38 results in increased periosteal osteogenesis. Coinciding with increased expression of these genes, we observed elevated levels of transcripts for Ihh and its target gene, Ptch, in response to p38 inhibition. Addition of recombinant hedgehog protein mimicked some effects of p38 inhibitors. We therefore suggest that p38 signaling regulates chondrocyte–perichondral cell communication during skeletal development, partially through increased Ihh signaling.

Introduction

During endochondral ossification, a cartilage intermediate, which is eventually replaced by mineralized bone, serves as a template for bone development [1], [2], [3]. The successful formation of endochondral bone demands a series of well-orchestrated events including chondrogenesis, chondrocyte maturation and hypertrophy, and finally matrix calcification and chondrocyte apoptosis. Upon condensation of mesenchymal cells, the central cells form the chondrocytes and the peripheral cells give rise to the perichondrium. Centrally positioned chondrocytes, after a period of proliferation, exit the cell cycle and commence differentiation into hypertrophic chondrocytes. The sequential proliferation and differentiation of chondrocytes establish a highly structured region of growth termed the growth plate that is situated between the metaphysis and the epiphysis of the long bone [4]. At the metaphyseal side of the growth plate, chondrocytes mineralize their surrounding matrix. In parallel with differentiation of the chondrocytes, perichondral cells on the periphery of the condensations develop into the bone collar or periosteum that provides for the vascularization of hypertrophic cartilage as well as for osteoblast precursor cells that will ultimately replace the chondrocytes [5]. The intricate control and differentiation of chondrocytes and perichondral cells within the growth plate region are therefore essential for normal bone growth.

Each of the cell types comprising bone expresses unique gene expression profiles that affect development and maturation of adjacent cell types. A classical example is Indian hedgehog (Ihh), which is expressed by prehypertrophic chondrocytes and together with PTHrP regulates the rate of chondrocyte differentiation in the growth plate [6]. Furthermore, Ihh expression by chondrocytes supports periosteal osteoblast differentiation [7]. While knowledge of such signaling proteins has gained ground, the intracellular signaling pathways controlling endochondral ossification are not completely understood. However, insight into some of these pathways has become apparent through analyses of the roles of mitogen-activated protein kinase (MAPK) pathways in skeletal cells in vitro. These studies have provided strong evidence that p38 MAPK plays a role in both chondrocyte [8], [9], [10], [11] and osteoblast [12], [13] differentiation. Our previous studies also support the requirement of p38 activity in the expression of hypertrophic chondrocyte markers such as collagen X [10], [11]. While many signals controlling the activity of p38 kinases have been described and are physiologically relevant in the context of the developing bone, such as bone morphogenetic proteins (BMPs) and transforming growth factor-β (TGF-β) [3], downstream targets are still largely undefined. Since control of target gene expression by p38 signaling appears to occur in a cell type-specific fashion [14], [15], identification of skeleton-specific targets will be essential for a comprehensive understanding of p38 function in bone tissues.

While chondrocytes and osteoblasts are widely regarded as related, but distinct cells, recent studies have shown that differentiating chondrocytes and osteoblasts share expression of many marker genes. Included in this repertoire are the Runt-related transcription factor 2 (Runx2), Osterix and (at least according to some studies) Osteocalcin, which were traditionally assumed to be confined to osteoblasts but are now accepted to be expressed by chondrocytes [16], [17], [18], [19], [20], [21]. Runx2 (also known as Cbfa-1) is a transcription factor whose expression in mice is first observed at embryonic day 9.5 in the notochord followed by prechondrogenic mesenchymal condensations [22] and then hypertrophic chondrocytes [18], [20]. While expression in the perichondrium/periosteum is noted by embryonic day 13.5, osteoblasts demonstrate the most significant levels of expression [23]. Knockout studies have shown that this transcription factor is essential for osteoblast formation and also plays an important role in terminal differentiation of chondrocytes [24].

Osterix is a zinc-finger containing transcription factor downstream of Runx2 and likewise is required for osteoblast differentiation and bone formation as demonstrated by failure of bone formation in Osterix-deficient mice [25]. Osterix expression has recently been shown in chondrocytes [21] where transcript levels are detected on embryonic day 13.5 of mice [25]. At this time, the surrounding perichondrium and the mesenchymal condensations of future membranous bones also show Osterix expression. By embryonic day 15, strong expression is found in cells associated with the trabeculae bone while expression in prehypertrophic chondrocytes of the growth plate is weak. At the time of birth, Osterix expression is restricted to cells of the bone matrix and the periosteum [25].

A number of important osteoblast genes are regulated by Osterix, including Osteocalcin, the major non-collagenous protein of the bone extracellular matrix. Transcription factors regulating Osteocalcin expression also include Runx2 [23], among others. In cartilage, expression of Osteocalcin by chondrocytes appears to be restricted to post-hypertrophic chondrocytes [16], [17], [26], but it should be noted that several studies failed to detect any Osteocalcin expression in chondrocytes at all [27], [28], [29].

In this study, we asked whether the p38 signaling pathway regulates expression of Runx2, Osterix and Osteocalcin in chondrocyte cultures and which mechanisms might mediate these effects. We demonstrate that p38 activity suppresses expression of osteoblast-associated genes in chondrocyte cultures. Our data suggest that elevated expression of osteoblast-associated genes in chondrocyte cultures in response to p38 inhibition occurs through induction of osteoblastic differentiation of perichondral cells. As Ihh has been shown to regulate chondrocyte proliferation, differentiation as well as osteoblast differentiation, we also examined a potential role of Ihh as a mediator of these effects.