Smurf2 induces degradation of GSK-3β and upregulates β-catenin in chondrocytes: A potential mechanism for Smurf2-induced degeneration of articular cartilage

Abstract

We have previously demonstrated that Smurf2 is highly expressed in human osteoarthritis (OA) tissue, and overexpression of Smurf2 under the control of the type II collagen promoter (Col2a1) induces an OA-like phenotype in aged Col2a1-Smurf2 transgenic mice, suggesting that Smurf2 is located upstream of a signal cascade which initiates OA development. However, the factors downstream of Smurf2 in this signal cascade and how Smurf2-induced OA is initiated are largely unknown. In this study, we further characterized the phenotypic changes in Col2a1-Smurf2 transgenic and WT articular cartilage from the postnatal stage to adulthood. We found that the articular cartilage degeneration occurring at the cartilage surface in 6 month-old Col2a1-Smurf2 transgenic mice progressed from an expanded hypertrophic domain in the basal layer of the deep articular cartilage at 2.5 weeks of age, which may lead to an accelerated calcification and ectopic ossification of this region at 1 month of age, and aggregation and maturation of articular chondrocytes in the middle and deep zones at 2 months and 4.5 months of age, respectively. Furthermore, we discovered that ectopically expressed Smurf2 interacted with GSK-3β and induced its ubiquitination and subsequent proteasomal degradation, and hence upregulated β-catenin in Col2a1-Smurf2 transgenic chondrocytes ex vivo. It is therefore likely that Smurf2-mediated upregulation of β-catenin through induction of proteasomal degradation of GSK-β in chondrocytes may activate articular chondrocyte maturation and associated alteration of gene expression, the early events of OA.

Introduction

The process of endochondral ossification consists of multiple stages. First, mesenchymal cells aggregate to form condensations, which subsequently differentiate into two types of cells: chondrocytes that form cartilage elements and osteoblast lineage cells that form the perichondrium surrounding the cartilage rudiment. Second, the differentiated chondrocytes undergo proliferation to form orderly parallel columns. The proliferating chondrocytes synthesize and deposit cartilage matrix such as type II collagen (Col2), and proteoglycans. Third, chondrocytes exit the cell cycle and mature into hypertrophic chondrocytes, and express the stage-specific marker type X collagen (ColX). Finally, terminally differentiated hypertrophic chondrocytes express matrix metalloproteinase (MMP) 13, and the cartilage matrix is degraded and replaced by bone.

Chondrocyte differentiation and maturation during endochondral ossification are tightly regulated by several key growth factors and transcription factors. Members of the TGF-β superfamily such as TGF-β isoforms and BMPs play important roles in the regulation of chondrocyte differentiation and maturation during this process. For example, TGF-β induces chondrogenesis including chondrocyte differentiation and cartilage matrix synthesis in developing limb buds [1], [2]. On the other hand, TGF-β signaling inhibits chondrocyte maturation and ColX expression during long bone lengthening, evidenced by loss of TGF-β signaling in the growth plate resulting in increased thickness of hypertrophic zone and upregulation of ColX expression [3], [4]. Similar to the stimulatory role of TGF-β signaling in chondrogenesis, BMP signaling is critical for mesenchymal condensation and chondrocyte differentiation during limb bud development [5], [6], [7], [8], [9]. However, in contrast to the inhibitory effect of TGF-β signaling on chondrocyte maturation, BMP signaling promotes this process [6], [10], [11]. TGF-β/BMP signaling is initiated by binding of these ligands to their receptors, which induces receptor phosphorylation and phosphorylation of receptor regulated Smads including Smad1, Smad5, Smad8 (BMP receptor), Smad2, and Smad3 (TGF-β receptor) [12], [13], [14], [15]. Recently, accumulating evidence suggests that canonical Wnt signaling, which is transduced via β-catenin, is implicated in multiple-steps of endochondral bone formation [16], [17], [18], [19], [20]. For example, ectopic β-catenin in cells of the chondrogenic lineage inhibits chondrocyte differentiation but stimulates chondrocyte maturation and ossification during embryonic development [16], [20].

In general, β-catenin levels are upregulated by Wnt ligands. β-catenin levels are normally limited through continuous proteasome-mediated degradation of phosphorylated β-catenin, which is catalyzed by the enzyme GSK-3β in a “destruction complex” brought together by Axin and APC [21], [22]. Upon Wnt ligand binding to its receptors, the complex is disassociated, and the kinase activity of GSK-3β is suppressed, and hence non-phosphorylated β-catenin accumulates in the cytoplasm and translocates into the nucleus to activate expression of Wnt target genes [23]. The β-catenin protein levels are therefore tightly regulated by the Wnt-mediated activity of the “destruction complex”, and any mechanism by which normal β-catenin regulation is uncoupled from Wnt signaling control could result in dysregulation of osteoblast/chondrocyte function, leading to alterations in bone mass or degenerative joints [24], [25], [26]. However, our understanding of the abnormal regulation of β-catenin in these diseases and animal models remains incomplete.

Osteoarthritis (OA) is a degenerative joint disease, and its developmental process recapitulates many of the events that occur during endochondral ossification. The primary feature in OA-affected joints is articular cartilage degeneration. Unlike the cartilage in the developing growth plate, in which chondrocytes mature and matrix turns over rapidly, the articular cartilage is a permanent tissue with minimal turnover of its cells and matrix [27], [28], [29]. The function of articular cartilage that ensures the frictionless movement of joints depends on the unique architecture of the extracellular matrix and zonal articular chondrocytes. The matrix is a highly hydrated network of collagen fibrils embedded in a gel of negatively charged proteoglycan molecules. The articular chondrocytes, which are sparsely distributed in the matrix, are responsible for maintenance of the matrix. Articular cartilage is developed postnatally and cross-link maturation of the collagen network is completed by adulthood. Appearance of articular cartilage is at postnatal 2 weeks when a small domain of secondary ossification occurs in the center of the epiphysis, which separates the articular cartilage from the growth plate cartilage [9], [30]. At this stage, the deep zone of the newly formed articular cartilage is a growth plate-like tissue and contains a thin layer of enlarged chondrocytes expressing ColX, which may be associated with formation of the calcified zone seen in adult articular cartilage [31], [32], [33]. By 1 month of age, the secondary ossification center is expanded, and the zonal articular cartilage formation is completed [33]. Articular chondrocytes normally do not undergo maturation. However, during OA development, articular chondrocytes undergo maturation, and express maturational marker genes such as ColX [34], [35], [36], [37], and MMP-13 [37], [38], [39], [40], which in turn degrades matrix and leads to articular cartilage degeneration [39], [41], [42], [43]. As OA progresses, osteophytes formed via endochondral ossification are often seen at the margins of the joint and bridge the joint cavity. While OA is a leading cause of disability in older people [44], [45], the underlying molecular mechanism is largely unknown. Although loss of TGFβ/Smad3 signaling in skeletal cells via genetic approaches results in OA in mice such as in the dominant-negative TGFβ type II receptor transgenic mice and in the Smad3 KO mice [3], [4], or local administration/overexpression of TGF-β in the knee joints induces osteophyte formation [46], [47], [48], these events do not occur in humans with OA. Recently, several lines of evidence suggest a role for increased Wnt/β-catenin signaling in OA development. For example, mutations in FRZB, a soluble Wnt antagonist, are OA risk factors, and increase hip OA susceptibility in women [26], [49], [50], which are further confirmed by the phenotype of Frzb KO mice, which are more susceptible to chemically-induced OA [51]. Consistently, high β-catenin protein levels were detected in human OA chondrocytes, but not in normal articular cartilage [52], [53], [54], indicating that increased β-catenin signaling in articular chondrocytes may be responsible for a conversion of normal cells to OA cells. However, the upstream signaling pathway(s) that dictates upregulation of β-catenin in articular chondrocytes during OA initiation is not clear.

We have recently demonstrated that Smurf2 is highly expressed in human OA tissue, and that overexpression of Smurf2 under the control of the type II collagen promoter (Col2a1) induces OA in mice, suggesting that Smurf2 is located upstream of a signaling cascade that is responsible for OA initiation in humans [37]. In some cell lines, ectopically overexpressed Smurf2 directly interacts with Smad1 and Smad2, and indirectly interacts with the type I receptor of TGF-β and β-catenin via an adaptor Smad7 for ubiquitination and proteasomal degradation [55], [56], [57], [58]. However, ectopic Smurf2 in Col2a1-Smurf2 transgenic chondrocytes does not induce the proteasomal degradation of these known targets identified in vitro but induces that of exogenous TGF-β activated phosphorylated Smad3, and decreases associated TGF-β/Smad3 signaling ex vivo [37]. Notably, the basal level of phosphorylated Smad3 and TGF-β/Smad3 signaling is slightly decreased in resting Col2a1-Smurf2 transgenic chondrocytes versus resting WT cells [37]. Given that Smad3 KO mice and Col2a1-Smurf2 transgenic mice develop severe OA at a similar age [4], [37], the mild loss of TGF-β/Smad3 signaling in Col2a1-Smurf2 transgenic chondrocytes may not be the major mechanism underlying Smurf2-induced OA in the transgenic mice. In this study, we demonstrate that ectopic Smurf2 in Col2a1-Smurf2 transgenic chondrocytes targets basal GSK-3β for ubiquitination and proteasomal degradation and increases the basal levels of β-catenin protein and signaling within these cells ex vivo. This novel signal cascade may represent a predominant mechanism that activates articular chondrocyte maturation, a critical early event during OA development.