CaSR modulates proliferation of the superficial zone cells in temporomandibular joint cartilage via the PTHrP nuclear localization sequence

The superficial zone cells in mandibular condylar cartilage are proliferative. The present purpose was to delineate the relation of calcium‐sensing receptor (CaSR) and parathyroid hormone‐related peptide nuclear localization sequence (PTHrP87‐139), and their role in the proliferation behaviors of the superficial zone cells. A gain‐ and loss‐of‐function strategy were used in an in vitro fluid flow shear stress (FFSS) model and an in vivo bilateral elevation bite model which showed mandibular condylar cartilage thickening. CaSR and PTHrP87‐139 were modulated through treating the isolated superficial zone cells with activator/SiRNA and via deleting CaSR or parathyroid hormone‐related peptide (PTHrP) gene in mice with the promoter gene of proteoglycan 4 (Prg4‐CreERT2) in the tamoxifen‐inducible pattern with or without additional injection of Cinacalcet, the CaSR agonist, or PTHrP87‐139 peptide. FFSS stimulated CaSR and PTHrP expression, and accelerated proliferation of the Prg4‐expressing superficial zone cells, in which process CaSR acted as an up‐streamer of PTHrP. Proteoglycan 4 specific knockout of CaSR or PTHrP reduced the cartilage thickness, suppressed the proliferation and early differentiation of the superficial zone cells, and inhibited cartilage thickening and matrix production promoted by bilateral elevation bite. Injections of CaSR agonist Cinacalcet could not improve the phenotype caused by PTHrP mutation. Injections of PTHrP87‐139 peptide rescued the cartilage from knockout of CaSR gene. CaSR modulates proliferation of the superficial zone cells in mandibular condylar cartilage through activation of PTHrP nuclear localization sequence. Our data support the therapeutic target of CaSR in promoting PTHrP production in superficial zone cartilage.


Abstract
The superficial zone cells in mandibular condylar cartilage are proliferative.
The present purpose was to delineate the relation of calcium-sensing receptor (CaSR) and parathyroid hormone-related peptide nuclear localization sequence

| INTRODUCTION
The temporomandibular joint (TMJ) condylar cartilage is composed of superficial fibrous, proliferative, prehypertrophic, and hypertrophic layers. 1 Cells in different layers are at various stages of differentiation, therefore, behave differently. 2 Chondrocytes go through a proliferation program and then further differentiate into postmitotic, hypertrophic chondrocytes. Intracellular calcium signaling, one of the earliest responses of chondrocytes to physical stimuli, 3 takes a role in regulating chondrocytes' maturation. 4 Calcium-sensing receptor (CaSR), a member of the G protein-coupled receptor family, is embedded in plasma membrane and intracellularly in Golgi and endoplasmic reticulum of almost all types of cells including chondrocytes. 5,6 CaSR senses changes in calcium levels. 7 Activated by high levels of extracellular calcium which is often induced by loading, the CaSR signal promotes the mature chondrocytes to terminal differentiation. 8 The specific knockout of CaSR in the type-2 collagen (Col-2) expressing mature chondrocytes during the embryonic period inhibits the terminal differentiation of chondrocytes, resulting in bone shortening and dwarfism in mice. 6 In osteoarthritic cartilage, CaSR expression is upregulated, and chondrocyte terminal differentiation is accelerated. 9 Inhibition of CaSR in Col-2-Cre pattern is beneficial for attenuation of osteoarthritis progression. 9 The superficial zone cartilage that harbors proliferative cells plays a crucial role in determining the dynamic compressive properties of the cartilage. 10 CaSR has been reported to express in this zone, 11 however, whether and how CaSR modulates the functional behaviors of the superficial zone cells is intriguing.
Calcium not only mediates the expression of CaSR but also regulates the expression of mechano-induction of parathyroid hormone (PTH)-related peptide (PTHrP), 12 which keeps chondrocytes proliferating and delays their further differentiation. 13 The high extracellular calcium promotes expression of the genes related to chondrocyte differentiation, and such an action can be blunted by incubating cells with PTHrP. 14 Suppression of chondrocytes hypertrophy caused by the low calcium concentration is PTHrP dependent. 4 PTHrP is synthesized in perichondrial cells and chondrocytes at the ends of the growing bones in the growth plate. 13 In the superficial zone cartilage, PTHrP is localized in nucleus by sequence. Our data support the therapeutic target of CaSR in promoting PTHrP production in superficial zone cartilage.

K E Y W O R D S
calcium-sensing receptor, cartilage, chondrocytes, parathyroid hormone-related peptide, temporomandibular joint

Significance and Innovations
1. In addition to promoting terminal differentiation of the mature chondrocytes, calciumsensing receptor (CaSR) has a promotion effect on the proliferation of the Prg4 + superficial zone cells in cartilage or the chondrocytes progenitors. Such a proliferation promotion effect of CaSR on cartilage is parathyroid hormonerelated peptide (PTHrP) nuclear localization sequence dependent. 2. CaSR regulated PTHrP nuclear localization sequence in the Prg4 + superficial zone cells to control cell proliferation in bilateral anterior elevation biomechanically treated temporomandibular joint cartilage. CaSR-PTHrP nuclear localization sequence is the therapeutic potential in promoting cell proliferation in cartilage. In the IF staining, the tissue between the two white dotted lines is the full zone condyle cartilage, and above the yellow dotted line is the condyle superficial zone cartilage. Scale bar, 100 or 10 μm. n = 6. Values are represented as the mean with lower and upper limits of 95% CI. *p < .0001 or as specified.
its nucleolar targeting signal by linking with a nuclear localization sequence (NLS) within 87 to 139 regions termed PTHrP 87-139 . PTHrP 87-139 has been found to promote the proliferation of cartilage cells. 14 PTHrP is also localized in the secretory pathway that inhibits the hypertrophy of chondrocytes by binding to PTH/PTHrP receptor expressed by mature chondrocytes, which recognizes the N-terminal within 1 to 34 regions of the PTHrP molecules. 15 Unloading is associated with rapid changes in PTHrP expression and articular chondrocyte differentiation. 16 It has been reported that the CaSR antagonizes the PTH1 receptor (PTH1R) signaling to promote chondrocytes' terminal differentiation. 11 In the spontaneous osteoarthritic cartilage of guinea pigs, CaSR expression became upregulated in the superficial zone at the early stage of osteoarthritis, while the upregulation of PTHrP as revealed by immunoassay occurred when osteoarthritis developed and progressed. 5 In our previously publication 9 we noticed that injection of Cinacalcet, the agonist of CaSR, in the sham control rats reduced the number of hypertrophied cells in TMJ cartilage. However, the cells in superficial zone cartilage, including fibrous and proliferative layers, were not decreased. 9 Such a result reminded us that CaSR may play different roles in different layers of condylar cartilage. In cartilage with osteoarthritic lesions, there are not only degeneration but also proliferation responses, the latter compensating for the reduction of cells caused by the promoted cellular differentiation in the degenerated osteoarthritis (OA) cartilage. 17,18 Whether CaSR in superficial zone activate PTHrP expression under loading, and hereafter takes the role in mediating cell proliferative behaviors remains to be determined. During chewing, TMJ condylar cartilage suffers loadings derived from the contraction of the jaw-closing muscles, which can be altered by changing the dental occlusal relation. Previously, we developed a bilateral anterior elevation (BAE) animal model that promoted the proliferative activity of chondrocytes. 19,20 Lubricin is a secreted proteoglycan encoded by the Prg4 gene, which is specifically expressed in the superficial layer of the articular cartilage. 21,22 Lubricin/Prg4-positive cells have been reported to function as progenitors for the underlying layers of the articular cartilage, 23 and to be sensitive to mechanical loading and protective against the development of OA. 24 In this study, we took genetic and pharmacological approaches to delineate the role of CaSR and PTHrP in Prg4 expressing superficial zone cells in TMJ condylar cartilage of mice subjected to BAE. Additional injection of PTHrP 87-139 peptide to TMJs was conducted. An in vitro fluid flow shear stress (FFSS) model was also adopted. The purpose was to explore the relation of CaSR and PTHrP NLS and their role in the proliferation behaviors of the TMJ superficial zone cells.

| Experimental animals
Animal source information is provided in Supplemental Materials and Methods Section.
By crossing Rosa26/tdTomato (TdT) mice with Prg4-Cre ERT2 mice, six 6-week-old female TdT-Prg4 litters were generated, in which Prg4-expressing cells were labeled by TdT when TAM (0.1 mg/g of body weight) was injected for 5 days.
For details of genotyping and TAM induction, and the TMJ injection methods please refer to the Supplemental Materials and Methods Section. The timeline is presented in Figure S1. The application of BAE, methods of tissue preparation, histology and immunofluorescence (IF) staining, and the primary superficial chondrocyte culture, FFSS application, methods of SiRNA transfection, total protein extraction, western blotting, and RNA extraction and quantitative real-time PCR assays are provided in Supplemental Materials and Methods Section.

| Statistical analysis
All the data were retrieved from independent samples or observations. Values are presented as the mean and 95% confidence intervals (lower and upper limits). The statistical analysis was performed using SPSS software, version 11.0 (SPSS Inc). For details, please refer to the Supplemental Materials and Methods Section. were located in the superficial fifth of the TMJ condylar cartilage ( Figure S2). In mouse TMJ cartilage, the cellular zonal arrangement is not as distinct as that in rat TMJ. We alternatively separated the TMJ superficial zone, deep zone, and full zone cartilage from 3-week-old SD rats under a microscope. Hematoxylin-eosin (HE) staining showed that the average superficial zone cartilage thickness of SD rats was 169.38 μm, approximately one-fifth of full zone cartilage with an average thickness of 847.38 μm ( Figure S3A-D). Therefore, even though the mouse TMJ cartilage is not distinctively zonal arranged, the one-fifth thickness of the condylar cartilage was defined as superficial zone cartilage in this study.

| BAE upregulated CaSR and
PTHrP expression in the TMJ superficial zone cartilage BAE increased the number of chondrocytes and the thickness of condylar superficial zone cartilage as revealed by HE staining (Figure 1A,B). IF staining indicated that in the control groups, CaSR was expressed less in the superficial region versus the deep area, while the PTHrP was widely distributed in the whole cartilage. In the BAE groups, however, the number of CaSR-positive cells, PTHrP-positive cells, and the ratio of CaSR and PTHrP double-positive cells to CaSR-positive cells or to PTHrPpositive cells were all significantly increased in the superficial fifth cartilage ( Figure 1C,D).

| CaSR promoted proliferative responses of the TMJ superficial zone cartilage through PTHrP
Superficial zone cells were separated from TMJ condylar cartilage of 3-week-old rats and then were treated with FFSS. Western blot data revealed that FFSS treatment for 2 and 4 h increased CaSR and PTHrP protein expression versus blank control (Cont, without any stimulation). Meanwhile, the protein expression of Ki67, a marker of cell proliferation, and Ihh, a marker of early chondrocyte differentiation, were both increased. While the protein of Col-X, a marker of chondrocyte terminal differentiation, was not detectable, implying that the superficial zone cells used were early differentiated other than mature differentiated under the present FFSS treatment (Figure 2A).
To elucidate the relation between CaSR and PTHrP, we added SiRNA against CaSR or PTHrP, respectively, to inhibit the expression of CaSR or PTHrP with or without 16 dyne/cm 2 FFSS treatment for 2 h. We also used the CaSR agonist Cinacalcet or PTHrP 87-139 peptide in a culture medium to activate the CaSR or PTHrP nuclear localization sequence, accordingly. Western Blot data showed that inhibiting CaSR or PTHrP by SiRNA significantly reduced the expression of CaSR or PTHrP protein, while Cinacalcet or PTHrP 87-139 peptide increased the expression of CaSR or PTHrP protein ( Figure S4A,B). Western Blot data showed that inhibiting CaSR by SiRNA suppressed the upregulation impact of FFSS on the protein expression of not only CaSR ( Figure S4C), but also PTHrP, Ki67, and Ihh ( Figure 2B). While activating CaSR by Cinacalcet did not increase the protein expression of CaSR under FFSS stimulation, however, it further increased the FFSS promoted protein expression of PTHrP, Ki67, and Ihh ( Figure 2C). In contrast, SiRNA of PTHrP did not change the protein expression level of CaSR under FFSS stimulation, however, it suppressed the expression of PTHrP ( Figure S4D), and reduced the FFSS upregulated expression of Ki67 and Ihh. Impressively, adding PTHrP 87-139 peptide in the culture medium did not affect the expression of CaSR and PTHrP under FFSS stimulation, but promoted the FFSS-stimulated protein expression of Ki67 and Ihh ( Figure 2D). Under all the four conditions above, the protein expression level of Col-X was still undetectable (Figure 2A-D).
IF staining and Rt-PCR showed that after 16 dyne/cm 2 FFSS treatment for 2 h, the expression of aggrecan in the superficial zone chondrocytes was significantly increased. Inhibiting either CaSR or PTHrP via SiRNA suppressed the FFSS promoted expression of aggrecan, while Cinacalcet or PTHrP 87-139 peptide enhanced the promotion effect of FFSS on the expression of aggrecan ( Figure 2E-G). In the IF staining, the tissue between the two white dotted lines is the full zone condyle cartilage, and above the yellow dotted line is the condyle superficial zone cartilage. Scale bar, 100 μm. n = 6. Values are represented as the mean with lower and upper limits of 95% CI. *p < .0001 or as specified. The percentage in parentheses refers to the difference in the average value between the two groups.

| Both CaSR and PTHrP take a promotion role in BAE-stimulated TMJ superficial zone cartilage
To explore the role of CaSR in the superficial zone cartilage in vivo, we generated Prg4-Cre ERT2 ; CaSR flox/flox litters by crossing Prg4-Cre ERT2 mouse with CaSR flox/flox mouse. We have bred at least two generations and selected Prg4-Cre ERT2 ; CaSR flox/flox litters mice through gene identification. (Figure S5A,C). Continuing injection of TAM for 5 days was performed when the litters were 6 weeks old to knock down CaSR in Prg4 + tissue-specific pattern and obtain CaSR-KO littermates. BAE was also delivered at 6 weeks old (for the timeline, please refer to Figure S1). Littermates of CaSR flox/flox , TAM-treated CaSR flox/flox , and Prg4-Cre ERT2 ; CaSR flox/flox without injection of TAM were used as genetic controls, of which the phenotype was identical to that of wild type as we previously reported. 20 HE staining revealed that, in the CaSR-KO group, the cell number in TMJ cartilage and the cartilage thickness of the superficial zone were both reduced compared with that in the three genetic control groups. (Figure S6).
In the CaSR-KO group, the cell number in TMJ cartilage and the cartilage thickness of the superficial zone were both reduced compared with that in CaSR flox/flox group ( Figure 3A,B). IF data indicated that the CaSR-, PTHrP-, Ki67-, and Ihh-expressing cells in the superficial zone were all reduced in CaSR-KO group versus CaSR flox/flox group ( Figure 3C,D, Figure S7A-D). Similar regularity was noticed in BAE-treated littermates, which showed that the BAE promotion effect on cartilage was significantly suppressed (Figure 3A-D, Figure S7A-D).
We then generated Prg4-Cre ERT2 ; PTHrP flox/flox litters by crossing Prg4-Cre ERT2 mouse with PTHrP flox/flox mouse using the same methods ( Figure S5B,C). Continuing injection of TAM for 5 days was performed when the litters were 6 weeks old to knock down PTHrP in Prg4 + tissue-specific pattern and obtain PTHrP-KO littermates. BAE was also delivered at 6 weeks old (for the timeline, please refer to Figure S1). Littermates of PTHrP flox/flox , TAM-treated PTHrP flox/flox , and Prg4-Cre ERT2 ; PTHrP flox/flox without injection of TAM were used as controls. Our data showed that the phenotype in the three control groups was identical to that of wild type as we previously reported. 20 HE staining revealed that, in the PTHrP-KO group, the cell number in TMJ cartilage and the cartilage thickness of the superficial zone were both reduced compared with that in the three genetic control groups. (Figure S8).
In the PTHrP-KO group, the cell number in TMJ cartilage and the cartilage thickness of the superficial zone were both reduced compared with that in PTHrP flox/flox group ( Figure 4A,B). IF data indicated that in PTHrP-KO group, the PTHrP-, Ki67-, and Ihh-expressing cells in the superficial zone were all reduced compared with PTHrP flox/flox group ( Figure 4C,D, Figure S9A-D), while no such significant change in that of CaSR-expressing cells ( Figure 4C,D). Similar regularity was noticed in BAE-treated littermates, which showed that the BAE promotion effect on cartilage was significantly suppressed ( Figure 4A-D, Figure S9A-D).

| CaSR mediated BAE promotion impact on the superficial zone cartilage via PTHrP
In order to confirm the up and downstream relation between CaSR and PTHrP in superficial zone cartilage, we injected PTHrP 87-139 peptide to the TMJ region in CaSR-KO groups, taking the vehicle injection as a treatment control group. The results showed that injection of PTHrP 87-139 peptide increased the percentage of PTHrP-positive cells to total cells in superficial zone cartilage in CaSR-KO littermates with or without BAE treatment versus the vehicle injection groups, while that of CaSR-positive cells were not changed ( Figure S10A,B). The number of superficial zone condyle chondrocytes and the thickness of condyle superficial zone cartilage, and the number of Ki67-and Ihh-positive cells in superficial zone cartilage were all improved in CaSR-KO litters after injection of PTHrP 87-139 peptide with or without BAE ( Figure 5A-F).
We then injected the CaSR agonist Cinacalcet to the TMJ region in PTHrP-KO litters, taking the vehicle F I G U R E 4 PTHrP gene knockout (PTHrP-KO) inhibited the effect of bilateral anterior elevation (BAE) on condylar cartilage, but hardly impacted the CaSR expression. Superficial zone cartilage-specific and tamoxifen-inducible PTHrP-KO mice were used. The PTHrP flox/flox mice were used as the genetic control. BAE was applied to 6-week-old mice for 7 weeks. (A) Hematoxylin-eosin (HE) staining. (B) Comparisons of superficial zone Cho Number and superficial zone Car Thickness between the sham and BAE mice in the PTHrP flox/flox groups and PTHrP-KO groups. (C) Immunofluorescence (IF) staining of CaSR and PTHrP. (D) Comparisons of positive cell rate of CaSR and PTHrP in condylar superficial zone cartilage between the sham and BAE mice in the PTHrP flox/flox groups and PTHrP-KO groups. In the HE staining, the tissue above the black dotted line is the condyle superficial zone cartilage. In the IF staining, the tissue between the two white dotted lines is the full zone condyle cartilage, and above the yellow dotted line is the condyle superficial zone cartilage. Scale bar, 100 μm. n = 6. Values are represented as the mean with lower and upper limits of 95% CI. *p < .0001 or as specified. The percentage in parentheses refers to the difference in the average value between the two groups. In the HE staining, the tissue above the black dotted line is the condyle superficial zone cartilage. In the IF staining, the tissue between the two white dotted lines is the full zone condyle cartilage, and above the yellow dotted line is the condyle superficial zone cartilage. Scale bar, 100 μm. n = 6. Values are represented as the mean with lower and upper limits of 95% CI. of TMJ condylar cartilage, the percentage of Ki67-and Ihh-positive cells in condyle superficial zone cartilage with or without BAE. (Figure 6A-F).
Our data demonstrated the down-streamer relation of PTHrP to CaSR in BAE promoted superficial zone cartilage.

| DISCUSSION
In parathyroid glands, CaSR senses changes in serum calcium to regulate PTH. 25 PTHrP has been demonstrated to be produced in cells in superficial zone cartilage and functions as promoting cell proliferation; while CaSR has been reported to be expressed in mature chondrocytes and functions as promoting cell terminal differentiation. 7,9 Currently, we, for the first time, demonstrated, using the genetic in vivo modulation methods, that in Prg4-expressing superficial zone cells CaSR functions as an up-streamer of PTHrP. Ablating PTHrP in the Prg4expressing superficial zone cells suppressed the proliferation, but showed no impact on CaSR expression, and pharmacologically activation of CaSR in PTHrP ablation animals could not stimulate the proliferative responses. While ablating CaSR in the Prg4-expressing superficial zone cells suppressed PTHrP localization sequence and cell proliferation, pharmacologically activation of PTHrP NLS in CaSR ablation animals rescued the genetic suppressed proliferative responses. That means, CaSR, in addition to promoting cell differentiation in mature chondrocytes, 9 mediated the function of the biomechanically promoted PTHrP expression, hereafter taking the role in the proliferative behaviors of the superficial zone cells.
During the present in vitro experimental period, the expression of Col-X, the marker of chondrocytes mature differentiation was undetectable, implying a limited terminal differentiation of the cultured superficial zone cells under FFSS treatment. Literature has demonstrated that targeted deletion of Ihh results in short-limbed dwarfism, with decreased extensive hypertrophy in addition to decreasing chondrocyte proliferation. 27 We currently used Ihh as an indicator of superficial zone cell differentiation.
Based on the present and published data, it seems that CaSR can deliver different impacts on the cells in the cartilage at different differential stages via activating different downstream molecules, and the impacts of CasR on superficial zonal cartilage linked with PTHrP signal. The interplay between CaSR and PTH1R signaling has been reported. 9 Mediation of CaSR on the expression reduction in PTH1R, the target of the PTHrP protein 1-34 sequence (PTHrP 1-34 ), 28 was proposed as one of the mechanisms. 14 However, it has been demonstrated that the regulation of PTHrP on cell proliferation in superficial zone cartilage is conducted by PTHrP NLS which is the function of the PTHrP 87-139 other than PTH/PTHrP receptor signal. 14 The present data indicated the impact of CaSR on cell proliferation in the superficial zone cartilage via PTHrP production and the function of PTHrP nuclear localization sequence. That means, in the superficial zone cartilage, CaSR stimulates cell proliferation via promoting PTHrP production, thus PTHrP nuclear localization sequence. Such a variation in CaSR function provides a possible explanation, at least partially, for the unsatisfactory results of using Cinacalcet, CaSR agonist, such as that observed in treating osteoporosis. 29 Further studies related to the impact of PTHrP NLS on chondrocytes behaviors in vivo are expected to be done by modifying the use of the transgenic animal model which, unfortunately, had been reported early lethality. 14

| CONCLUSION
CaSR modulates proliferation and differentiation of the superficial zone cells in mandibular condylar cartilage through PTHrP nuclear localization sequence. Our present and published 9 data indicated that targeting CaSR in Col2-expressing cells will prevent cell loss via terminal differentiation while our current data indicating that targeting CaSR in Prg4-expressing cells will prevent cell proliferation via suppressing PTHrP nuclear localization sequence. We provided an explanation for the inconsistent results in literature regarding the effect of CaSR intervention on joint problems, and support the therapeutic potential of the clinically available Cinacalcet in promoting PTHrP secretion in TMJ superficial zone cartilage by targeting the CaSR.