*Article* **Discoidin Domain Receptors 1 Inhibition Alleviates Osteoarthritis via Enhancing Autophagy**

**Hsin-Chaio Chou 1,2,3, Chung-Hwan Chen 2,3,4,5,6, Liang-Yin Chou 1,2,3, Tsung-Lin Cheng 2,3,7, Lin Kang 8, Shu-Chun Chuang 2,3, Yi-Shan Lin 2,3, Mei-Ling Ho 1,2,3,7,9,10, Yan-Hsiung Wang 2,3,11, Sung-Yen Lin 1,2,3,4,6,9,\* and Chau-Zen Wang 1,2,3,7,12,\***


Received: 30 July 2020; Accepted: 16 September 2020; Published: 23 September 2020

**Abstract:** We recently reported that the chondrocyte-specific knockout of discoidin domain receptors 1 (Ddr1) delayed endochondral ossification (EO) in the growth plate by reducing the chondrocyte hypertrophic terminal differentiation, and apoptosis. The biologic and phenotypic changes in chondrocytes in the articular cartilage with osteoarthritis (OA) are similar to the phenomena observed in the process of EO. Additionally, autophagy can promote chondrocyte survival and prevent articular cartilage from degradation in OA. On this basis, we explored the effect of Ddr1 inhibition on OA prevention and further investigated the roles of autophagy in treating OA with a Ddr1 inhibitor (7 rh). The anterior cruciate ligament transection (ACLT)–OA model was used to investigate the role of 7 rh in vivo. Forty 8-week-old mice were randomly assigned to four groups, including the sham group, ACLT group, and two treated groups (ACLT with 7 rh 6.9 nM or 13.8 nM). According to the study design, normal saline or 7 rh were intra-articular (IA) injected into studied knees 3 times per week for 2 weeks and then once per week for 4 weeks. The results showed that 7 rh treatment significantly improved the functional performances (the weight-bearing ability and the running endurance), decreased cartilage degradation, and also reduced the terminal differentiation markers (collagen type X, Indian hedgehog, and matrix metalloproteinase 13). Moreover, 7 rh decreased chondrocyte apoptosis by regulating chondrocyte autophagy through reducing the expression of the mammalian target of rapamycin and enhancing the light chain 3 and beclin-1 expression. These results demonstrated that the IA injection of 7 rh could reduce the chondrocyte apoptosis and promote chondrocyte autophagy, leading to the attenuation of cartilage degradation. Our observations

suggested that the IA injection of 7 rh could represent a potential disease-modifying therapy to prevention OA progression.

**Keywords:** discoidin domain receptors 1 (Ddr1); osteoarthritis (OA); autophagy; apoptosis; terminal differentiation

#### **1. Introduction**

Osteoarthritis (OA) is the most prevalent joint disease worldwide and also the leading cause of disability in aged patients. Because of aging and increasing obesity in the population, the prevalence of OA is rapidly increased. The current pharmacological therapy for OA was focused on symptomatic relief. Despite some promising disease-modifying therapy undergoing clinical trials, there is no currently effective therapy to prevent the progressive destruction of articular cartilage [1].

Chondrocyte death is the key pathogenesis of OA. The articular chondrocyte is the only resident cell in cartilage and is responsible for maintaining the equilibrium of the extracellular matrix. The compromising of chondrocyte survival leads to cartilage degradation; as a result, the prevention of chondrocyte death is an important research direction for OA prevention. The morphologic changes in chondrocytes in OA are resembling those happening in the growth plates, where the chondrocytes undergo hypertrophic terminal differentiation, featuring chondrocyte hypertrophic change, mineralization, and eventually apoptosis [2]. Recent research on parathyroid hormone 1-34 (PTH 1-34) in OA has indicated that the treatment can inhibit the apoptosis of chondrocytes in the growth plate, which can also be used to inhibit the apoptosis of chondrocyte in osteoarthritic articular cartilage, thereby reducing cartilage degradation [3–5].

Discoidin domain receptors (Ddr) are a group of the transmembrane receptors that triggers a ligand-induced kinase activation and receptor autophosphorylation after biding collagen and then to regulate cell migration, proliferation, differentiation, and cell survival [6]. Ddr1 and Ddr2 are two major types of Ddrs with similar amino acid sequences and both are expressed in the cartilage. The biological function of Ddrs has been widely investigated by gene knockout mice. Although the physiologic role of two Ddrs is different, the global Ddr1 or Ddr2 ablated mice all present with dwarfism [7]. The Ddr2 ablation reduces the chondrocyte proliferation and thus decreases bone growth; however, the reasons for dwarfism in Ddr1 ablation mice remain unclear. Our recent study using chondrocyte-specific Ddr1 knockout mice demonstrated that the Ddr1 ablation resulted in a decreased chondrocyte proliferation, terminal differentiation, and apoptosis in growth plates and caused a delayed EO [8]. Our findings indicated that Ddr1 is an imperative regulator of EO in the growth plate and maybe also a potential target for OA treatment.

Autophagy maintains the homeostatic equilibrium to cartilage following injury and is reported to be a cellular self-protection mechanism of normal cartilage. Previous researchers demonstrated that the deregulation of autophagy predisposed to the pathogenesis of OA and the activation of autophagy could protect articular cartilage from OA [9]. The mammalian target of rapamycin (mTOR) is a serine/threonine protein kinase regulating signal transduction during autophagy. A previous study showed that the cartilage-specific deletion of mTOR upregulated the autophagy signaling and reduced the articular degradation, chondrocyte apoptosis, and synovial fibrosis in surgical-induced OA mice [10]. Similarly, rapamycin (an inhibitor of mTOR) enhanced the activation of autophagy, maintained the cartilage cellularity, and reduced the severity of surgical-induced OA [11]. Although we do not fully understand the relationship between chondrocyte survival and autophagy, accumulating evidence suggests that the restoration autophagy of senescent chondrocyte can restore the reparative capacity and may be an effective therapeutic approach for OA [12].

While the inhibiting of Ddr1 can decrease chondrocyte apoptosis in the growth plate, it has yet to be demonstrated whether this phenomenon may occur in the articular cartilage and the inhibition expression of Ddr1 can protect against cartilage degradation in experimental-induced OA animal models. Ddr1 inhibition can reduce chondrocyte terminal differentiation and apoptosis in the growth plate, but whether autophagy contributes to the treatment of Ddr1 suppression on ameliorating OA progression remains unclear. The object of this study was to test the hypothesis that the inhibition of Ddr1 expression by intra-articular (IA) injection of Ddr1 inhibitor (7 rh) can reduce the OA progression in anterior cruciate ligament transection (ACLT)-induced OA mice. Specifically, we aimed to determine whether (1) IA of 7 rh can delay the articular cartilage degradation in histology and also improve knee function after OA induction; (2) IA of 7 rh can reduce the chondrocyte terminal differentiation and apoptosis; (3) the effects of 7 rh on prevention chondrocyte apoptosis are through the regulation of autophagy in chondrocytes.
