Knockdown of mechanosensitive adaptor Hic-5 ameliorates post-traumatic osteoarthritis in rats through repression of MMP-13

Osteoarthritis (OA) is the most common joint disease associated with articular cartilage destruction. Matrix metalloproteinase-13 (MMP-13) has an essential role in OA pathogenesis by degradation of collagen II, a major component of articular cartilage. Hydrogen peroxide-inducible clone-5 (Hic-5; TGFB1I1), a transforming growth factor-β-inducible mechanosensor, has previously been reported to promote OA pathogenesis by upregulating MMP-13 expression in mouse osteoarthritic lesions. In our current study, immunohistochemical analysis showed that Hic-5 protein expression was increased in human OA cartilage compared with normal cartilage. Functional experiments demonstrated that Hic-5 and MMP-13 expression was increased by mechanical stress, and mechanical stress-induced MMP-13 expression was suppressed by Hic-5 siRNA in human chondrocytes. Moreover, intracellular localization of Hic-5 shifted to the nucleus from focal adhesions in human chondrocytes subjected to mechanical stress, and nuclear Hic-5 increased MMP-13 gene expression. In vivo, intra-articular injection of Hic-5 siRNA decreased the Osteoarthritis Research Society International score and MMP-13 protein expression in articular cartilage of OA rats. Our findings suggest that Hic-5 regulates transcription of MMP-13 in human chondrocytes, and Hic-5 may be a novel therapeutic target for OA because OA progression was suppressed by intra-articular injection of Hic-5 siRNA in rats.


Introduction
Osteoarthritis (OA) is the most prevalent joint disorder characterized by articular cartilage degradation.6][7] MMP-13 knockout in mice protects against cartilage degradation caused by surgical induction compared with wildtype (WT) mice. 8Conversely, transgenic mice with constitutively active MMP-13 expressed speci cally in cartilage show articular cartilage erosion similar to human OA. 9 Hydrogen peroxide-inducible clone-5 (Hic-5) is a scaffold protein isolated as a gene induced by hydrogen peroxide and transforming growth factor β (TGF-β). 10We have previously demonstrated that subcellular localization of Hic-5 shifts from focal adhesions to stress actin bers in response to mechanical stress and Hic-5 controls the contractile capability of the cell. 11Furthermore, Hic-5 has been reported to be involved in the pathogenesis of various disorders.Our previous study showed that Hic-5 increases activated MMP-2 by regulating the expression of membrane type-1 MMP, which leads to the formation of abdominal aortic aneurysms and rupture. 12In breast tumors, Hic-5 is required for extracellular matrix (ECM) deposition and cell contractility, and metastasis to the lungs decreases in Hic-5-de cient mice. 13though there are differences in the detailed mechanisms of Hic-5 involvement in these disorders, ECM remodeling is a common mechanism.
Recently, we found that Hic-5 expression increases in mouse articular cartilage during early OA development, and mice lacking Hic-5 have signi cantly less cartilage erosion than WT mice. 14In vitro experiments using murine chondrocytes also demonstrated that Hic-5 de ciency decreases MMP-13 expression induced by excessive mechanical stress.This study aimed to determine whether Hic-5 is involved in MMP-13 expression induced by excessive mechanical stress in human chondrocyte, and to investigate the e cacy of Hic-5 as a therapeutic target for OA in vivo.

Signi cant increase in Hic-5 expression in human OA cartilage
To explore the role of Hic-5 in human OA pathogenesis, we rst examined Hic-5 expression in human OA cartilage.Immunohistochemistry showed that Hic-5 expression was higher in OA cartilage than in normal cartilage (Fig. 1, A and B), and the number of Hic-5-positive cells was signi cantly increased in OA cartilage (Fig. 1C).These results were consistent with our previous report indicating that Hic-5 was highly expressed in the cartilage of mice with surgically induced OA. 14

Suppression Of Mmp-13 Expression Induced By Mechanical Stress Following Hic-5 Knockdown In Human Chondrocytes
In the previous study, we demonstrated that Hic-5 and MMP-13 expression in mouse articular chondrocytes was increased by excessive mechanical stress. 14Therefore, we investigated whether human articular chondrocytes also had increased expression of Hic-5 and MMP-13 induced by mechanical stress in addition to other MMPs and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1).
As a result, Hic-5, MMP-3, and MMP-13 mRNA levels were increased in human chondrocytes stimulated by mechanical stress compared with unstimulated chondrocytes (Fig. 2A).MMP-1 expression tended to be upregulated by mechanical stress, but the difference was not signi cant, and there was no effect of mechanical stress on MMP-2 or TIMP-1 expression.Next, we examined the effect of Hic-5 knockdown on MMP gene expression using Hic-5 small interfering RNA (siRNA) and control siRNA as negative control.
Although Hic-5 knockdown by siRNA did not alter MMP-13 expression without mechanical stress, mechanical stress-induced MMP-13 expression was signi cantly reduced by Hic-5 siRNA compared with control siRNA.However, Hic-5 siRNA had no effect on MMP-1 or MMP-3 expression (Fig. 2B).These results were similar to our previous results observed in chondrocytes isolated from Hic-5 knockout mice, indicating that Hic-5 speci cally regulated MMP-13 gene expression among other MMPs under mechanical stress.

Translocation Of Hic-5 From Focal Adhesions To The Nucleus In Response To Mechanical Stress
We next observed intracellular localization of Hic-5 in human chondrocytes with or without mechanical stress.Hic-5 was detected at focal adhesions under unstimulated conditions, whereas Hic-5 expression in focal adhesions was attenuated after 0.5 Hz, 10% cyclic tensile strain loading for 30 minutes (Fig. 3A).Moreover, Hic-5 staining was prominent in the nucleus after cyclic tensile strain loading for 3 hours (Fig. 3A).Hic-5 normally shuttles between focal adhesions and the nucleus via a nuclear export signal (NES). 19Considering the possibility that mechanical stress accelerates the shuttle velocity, we examined changes in the subcellular localization of Hic-5 after cyclic tensile strain under treatment with leptomycin B (LMB), an NES inhibitor.The signal intensity in the nucleus was slightly increased by LMB at 1 hour after treatment, whereas the addition of both mechanical stress for 1 hour and LMB clearly induced nuclear localization of Hic-5 in human chondrocytes (Fig. 3B).Our previous study reported that nucleusaccumulated Hic-5 in response to H 2 O 2 participates in transcriptional control of c-fos. 19Taken together, these data imply that translocation of Hic-5 from focal adhesions to nucleus caused by mechanical force regulated MMP-13 expression.

In vivo knockdown of Hic-5 suppresses the progression of surgically induced OA in rats
We evaluated the therapeutic potential of Hic-5 in OA development using a rat surgical model of OA.First, we designed rat Hic-5 siRNA and validated the effect in both JTC-19 and RAT-2 rat cell lines.Hic-5 expression was remarkably suppressed in Hic-5 siRNA-expressing cells compared with the control (Fig. 5A).Next, Hic-5 siRNA was injected into the intra-articular spaces of rat knee joints every 3 days from day 10 to 19 after medial meniscectomy (MMx) (Fig. 5B).
Histological analysis showed that the formation of OA lesions had already occurred at 10 days postoperatively.At day 19, which was 9 days after the start of siRNA injection, the siRNA-injected rat group showed inhibited OA progression and lower Osteoarthritis Research Society International (OARSI) scores than the vehicle group (Fig. 5, C, D and Supplemental Table .2).Additionally, immunohistochemistry con rmed a decrease in Hic-5 and MMP-13 expression in knee cartilage from the siRNA-injected group compared with groups without siRNA injection, including day 10, untreated, and vehicle groups (Fig. 6, A, B and Supplemental Fig. 1).Taken together, these results indicated that Hic-5 induced by excessive mechanical stress enhanced OA development by increasing MMP-13 transcription (Fig. 6C).

Discussion
The current study characterizes Hic-5 as a major regulator of OA development by promoting cartilage degradation.There was a signi cant increase in Hic-5 expression in human OA cartilage, but not in non-OA cartilage.In vitro experiments showed that excessive mechanical stress induced Hic-5 and MMP-13 expression in human chondrocytes, and Hic-5 knockdown suppressed the elevated expression of MMP-13, the major ECM-degrading enzyme in OA formation.Additionally, excessive mechanical stress altered the subcellular localization of Hic-5 from focal adhesions to the nucleus, and nuclear accumulation of Hic-5 resulted in the transcriptional regulation of MMP-13.In vivo experiments showed that intra-articular administration of Hic-5 siRNA downregulated MMP-13 expression and had a protective effect against cartilage degradation in OA model rats.Taken together, these results indicated that Hic-5 might be a promising therapeutic target for OA.
1][22] We previously reported that Hic-5 expression was higher in patients with these disorders than healthy controls.In liver and pancreatic brosis, Hic-5 acts as a scaffold for the TGFβ/Smad2 pathway and its de ciency signi cantly attenuates mouse liver and pancreatic brosis by a reduction of collagen production, which is the major ECM component in brosis. 20,21 urthermore, we demonstrated that overexpression of Hic-5 using an adenovirus vector in human normal broblasts increased the expression of lysyl oxidase (LOX) that increases ECM stiffness and enhances tumor progression. 22Even more intriguingly, azoxymethane-induced colorectal tumor incidence was suppressed in Hic-5-de cient mice compared with WT mice.In this study, we similarly found that Hic-5 regulated dissolution of ECM rich cartilage as a regulator of MMP-13 and that knockdown of Hic-5 by siRNA resulted in attenuation of surgically induced OA in rats.Additionally, Hic-5 expression was elevated in human OA cartilage.Taken together, Hic-5 may have a role as an ECM regulator in the process of human OA.
This study revealed the novel nding that mechanical stress induced Hic-5 gene expression and translocation in human chondrocytes.Hic-5 is located predominantly in focal adhesions and found in the nucleus under stimulation by ROS in normal human broblasts, and by TGF-β in normal human dermal broblasts without mechanical stress. 19,23 owever, we previously showed that Hic-5 translocated from focal adhesions to actin bers after mechanical stress in mouse embryonic broblasts. 11These results and the present data differ in terms of Hic-5 translocation, which may be due to the different mechanical stress conditions and different cell types used in the analysis.
In colorectal cancer, nucleus-accumulated Hic-5 induces expression of LOX that catalyzes crosslinking of collagen bers to increase ECM stiffness. 22Furthermore, Hic-5 translocates into the nucleus after mechanical stress and is involved in gene expression of matrix-degrading enzymes in chondrocytes.Stimulation to focal adhesion plaques by increased ECM stiffness is consistent with a mechanical stressloading condition.Taken together, Hic-5 is likely to play a reciprocal role in regulating ECM microenvironmental rigidity by sensing an increase in ECM stiffness, which leads to nuclear translocation and induction of gene expression related to ECM rigidity control.Although Hic-5 gene expression was slightly increased by mechanical stress, MMP-13 expression was signi cantly suppressed in human chondrocytes.These results imply that the nuclear translocation of Hic-5 rather than increase in its expression induced by mechanical stress is important for OA development.
Recently, various studies of animal OA models treated by intra-articular injection of small molecule drugs have been performed to explore the potential of novel therapeutic medicines.5][26][27][28][29] Hic-5 has been conventionally considered di cult to inhibit by small molecular drugs because it is an intracellular adaptor protein and has no enzymatic activity.
However, inhibition of Hic-5 in vivo has been recently made possible by nucleic acid therapeutics, a new tool capable of selective gene knockdown across plasma membranes.In the present study, intra-articular injection of Hic-5 siRNA suppressed OA progression in rats.Thus, we not only identi ed Hic-5 as a therapeutic target for OA, but also established that Hic-5 siRNA may be novel therapeutic medicine for OA.
In summary, we demonstrated that Hic-5 regulates cartilage degradation as a transcriptional mediator of MMP-13.Our ndings suggest that Hic-5 siRNA has potential therapeutic application, which may be clinically useful in OA.

Immuno uorescence
Formalin-xed, para n-embedded human articular cartilage tissue sections were purchased from ORIGENE (MD, USA).Rat tibial cartilage was xed in 10% buffered formalin, decalci ed in formic acid, embedded in para n, and cut into 4-µm-thick sections.
For immunocytochemistry, cultured chondrocytes were xed in 3.7% buffered formalin and blocked with 3% bovine serum albumin (Sigma Aldrich, Taufkirchen, Germany)/phosphate buffered saline (PBS) containing 0.1% Tween-20.Cells were stained with the primary antibody at room temperature for 1 hour and then incubated with Alexa Fluor-conjugated secondary antibodies (Invitrogen, MA, USA).
Target gene expression was normalized to GAPDH using the 2 −ΔΔCt method.

Rat Oa Model
Animal experiments were approved by the Animal Care and Use Committee of UNITECH Co. Ltd. and conducted at UNITECH in accordance with the ethical guidelines.All methods were reported in accordance with ARRIVE guidelines (https://arriveguidelines.org).Seven-week-old Slc:Wistar male rats were housed under a 12-h light cycle in a temperature-controlled room for 1 week before surgery was performed.Experimental OA was induced by MMx. 16Brie y, anesthesia was induced in rats (medetomidine (2 mg/kg bodyweight), midazolam (0.4 mg/kg), butorphanol (5 mg/kg)).The knees and surrounding areas were shaved.A longitudinal incision was made on the anterior aspect of the right knee.Then, we transected the medial collateral ligament and anterior cruciate ligament of the right knee.Next, the medial meniscus was removed.The knee capsule and subcutaneous tissue were sutured and the skin was closed.Intra-articular treatment was initiated 10 days after surgery.Either Hic-5 siRNA (forward: 5 -GGAUCAUCUAUACAGCACA-3 ; reverse: 5′-UGUGCUGUAUAGAUGAUCC-3′, 10 nmol/L) (n = 8) or nucleasefree water (n = 8) as the vehicle with AteloGene Local Use Quick gelation (Koken, Tokyo, Japan) was injected into the intra-articular spaces of rat knee joints in accordance with the manufacturer's protocol.
OA severity was quanti ed using the OARSI scoring system by UNITECH Co. Ltd. 17,18 Statistical analysis Data normality was assessed by the Shapiro-Wilk normality test.When the distribution was normal, the unpaired t-test was used to compare two groups of samples and one-way analysis of variance with Tukey's multiple comparisons test was used to compare data from more than three groups.The Kruskal-Wallis test followed by Dunn's test was used to compare nonparametric data from multiple groups.All analyses were performed with GraphPad Prism software.Results are reported as the mean ± SEM.Pvalues of less than 0.05 were considered signi cant.

Figure 5 In
Figure 5