Regulatory Mechanisms of the Ihh/PTHrP Signaling Pathway in Fibrochondrocytes in Entheses of Pig Achilles Tendon

This study is aimed at exploring the effect of stress stimulation on the proliferation and differentiation of fibrochondrocytes in entheses mediated via the Indian hedgehog (Ihh)/parathyroid hormone-related protein (PTHrP) signaling pathway. Differential stress stimulation on fibrochondrocytes in entheses was imposed. Gene expression and protein levels of signaling molecules including collagen type I (Col I), Col II, Col X, Ihh, and PTHrP in the cytoplasm of fibrochondrocytes were detected. Ihh signal blocking group was set up using Ihh signaling pathway-specific blocking agent cyclopamine. PTHrP enhancement group was set up using PTHrP reagent. Ihh/PTHrP double intervention group, as well as control group, was included to study the regulatory mechanisms of the Ihh/PTHrP signaling pathway in fibrochondrocytes. Under low cyclic stress tensile (CTS), PTHrP, Col I, and Col II gene expression and protein synthesis increased. Under high CTS, Ihh and Col X gene expression and protein synthesis increased. Blocking Ihh signaling with cyclopamine resulted in reduced PTHrP gene expression and protein synthesis and increased Col X gene expression and protein synthesis. Ihh and PTHrP coregulate fibrochondrocyte proliferation and differentiation in entheses through negative feedback regulation. Fibrochondrocyte is affected by the CTS. This phenomenon is regulated by stress stimulation through the Ihh/PTHrP signaling pathway.


Introduction
Entheses damage and degeneration are common motor system diseases, with no ideal treatments at present [1]. The organizational structure of entheses has four layers, of which the fibrous cartilaginous layer plays an important role in entheses development and degeneration [2]. The development of entheses is associated with the proliferation and differentiation of fibrochondrocytes.
Stress stimulation is a major influencing factor for entheses development and degeneration [3][4][5][6][7]. Stress stimulation affects the balance of extracellular matrix synthesis and decomposition of chondrocytes, and hence changes the metabolism of articular chondrocytes. Multiple cytokines participate in the proliferation and differentiation of chondrocytes in entheses. Among these, stress-related protein parathyroid hormone-related protein (PTHrP) is known to promote chondrocyte proliferation and cartilage layer development [8][9][10]. Indian hedgehog (Ihh) is an upstream molecule of PTHrP [11]. Ihh is an important regulatory molecule during chondrocyte proliferation and differentiation. PTHrP is an indispensable limiting regulatory factor for chondrocyte proliferation and differentiation and controlled mainly by its upstream secretory protein Ihh. Ihh and PTHrP signals together maintain the dynamic equilibrium of chondrocyte development and metabolism [12,13]. However, how stress stimulation mediates Ihh/PTHrP signaling pathway to regulate chondrocyte proliferation and differentiation in entheses is largely unknown.
Therefore, it is worthwhile to study how stress stimulation mediates Ihh/PTHrP signaling pathway to regulate chondrocyte proliferation and differentiation in entheses, which has an extensive application value in clinic.
Cyclopamine was centrifuged at 1000 rpm for 5 min, and 200 L of dimethyl sulfoxide was added to dissolve cyclopamine completely. Then, 240 mL of Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS) and 1% antibiotics was added to rinse the reagent bottle 5 times.

PTHrP (Final Concentration = 10 nM).
Human PTHrP was purchased from Peprotech (cat# 100-09 50 g, lot# 1002267, L122). PTHrP was centrifuged at 1000 rpm for 5 min. Then, 5 mL of DMEM with 10% FBS and 1% antibiotics was added to wash the reagent bottle five times and stored in 10 mL centrifuge tubes. PTHrP (1 mL) was added into 99 mL of DMEM (with 10% FBS and 1% antibiotics). The remaining reagents in the centrifuge tubes were stored at 8 ∘ C.
All secondary antibodies were from Zhongshan Co. (Beijing, China).

Cell
Culture. The fibrochondrocytes were collected by two-step enzyme digestion [14]. We separated and cut the calcified fibrocartilaginous layer and Fibrocartilage tissue was chopped into pieces then placed in a digestion chamber. After incubation with 0.2% trypsin for 2 h and 0.2% collagenase for 18 h (GIBCO, Invitrogen Inc., Carlsbad, CA, USA), fibrocartilaginous cells were centrifuged, and the pellet was resuspended in Dulbecco's modified eagle serum (DMEM) (GIBCO, Invitrogen Inc., Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS) (GIBCO, Invitrogen Inc., Carlsbad, CA, USA) and 1% penicillin/streptomycin (GIBCO, Invitrogen Inc., Carlsbad, CA, USA). Cells were seeded into a T-25 vented flask, were grown to 80-90% confluence, and were used at the third passage. Fibrochondrocytes at the third passage were characterized using hematoxylin-eosin (HE) staining, alcian blue staining (indicative of glycosaminoglycan), and types I and II collagen immunohistochemistry. After identification, the culture cells were passaged to the second generation and plated at a density of 2 × 10 5 in a Bioflex six-well plate coated with Col I. The cells were incubated in a 37 ∘ C constant-temperature incubator with 5% CO 2 . The media was changed the next day. The cells were cultured until the six-well plate was overspread with 85%-90% cells.

Control
Group. DMEM with 10% FBS + 1% antibiotics was added into four 200 mL flasks (10 mL each) and incubated in a 37 ∘ C incubator with 5% CO 2 , for 3, 6, 12, or 48 h. in the body and undergo specific biochemical changes to respond and adapt to deformation.

Statistics.
The data were analyzed using SPSS 16.0 (SPSS, IL, USA). Each test was repeated three times under the same conditions to get average data as mean ± standard error of means. Statistical analysis of all parameters was performed using analysis of variance and Tukey test. A value less than 0.05 was considered statistically significant.

Results
After inhibition of Ihh signaling upstream of PTHrP by adding cyclopamine, PTHrP gene expression decreased significantly over time ( < 0.05) (Figure 2(a)). The relative values of PTHrP expression in the intervention groups versus the control group were 0.75 ± 0.08, 0.54 ± 0.10, 0.32 ± 0.12, and 0.12 ± 0.06 after 3, 6, 12, and 24 h, respectively (Table 3). Meanwhile, Col I and Col II expression decreased over time as well ( < 0.05) (Figure 1 respectively. Col X expression, however, increased over time ( < 0.05) (Figure 1(a)). The relative gene expression of Col X increased from 1.39 ± 0.21 to 3.61 ± 0.29 (from 3 to 24 h of Ihh inhibition) ( Table 1). Col I, Col II, and Col X protein synthesis under different intervention conditions was displayed in Table 2. Ihh and PTHrP protein synthesis under different intervention conditions was displayed in Table 4. Western blot of Col I, Col II, and Col X protein expression under different intervention conditions was displayed in Supplementary Figure 1 Supplementary Figures 2-6.
In CTS experiments, using a 4%, 8%, 12% strain load at 1 Hz, types I, II, and X collagen mRNA expression and protein secretion increased with increased exposure time ( < 0.05) (Figure 4). Thus, results show that appropriate mechanical stimulation promoted tendon development by the secretion of types I and II collagen, while excessive mechanical stimulation increased the degeneration of tendon tissue into mineralized tissue.
In CTS experiments, using a 4%, 8% strain load at 1 Hz, PTHrP and Ihh mRNA expression and protein secretion increased with increased duration of strain load (Figures 3(a), 3(b), 3(d), and 3(e)). But at 12% strain load at 1 Hz, PTHrP and Ihh mRNA expression and protein secretion reduced with increased time of strain load. These results suggest that mechanical stimulation affected PTHrP secretion.

Discussion
Chondrocyte proliferation and differentiation are controlled by a series of growth factors and endocrine hormones, of Stem Cells International 5       which Ihh/PTHrP signaling pathway plays the most important regulatory role [15][16][17]. During cartilaginous osteogenesis, the main role of PTHrP is to promote chondrocyte proliferation. Meanwhile, PTHrP inhibits chondrocyte hypertrophy, to inhibit chondrocyte differentiation and maturation. Ihh/PTHrP signaling pathway is an important but complex regulatory pathway to control cartilage development.
It has been proved that PTHrP stimulates chondrocyte proliferation but inhibits chondrocyte maturation, hence prolonging the cartilaginous osteogenesis, and helps in the formation of the complex skeleton shape and structure. Chondrocyte proliferation and differentiation were more rapid in PTHrP gene knockout mice than in wild-type mice. Also, the terminal differentiation of chondrocytes was faster, while the accretion zone at the cartilage growth plate shortened significantly [10]. In contrast, overexpression of PTHrP or continuous activation of parathyroid hormone 1 receptor resulted in the inhibition of chondrocyte differentiation and hypertrophy. PTHrP downregulates bone morphogenetic protein (BMP) signaling through RunX2, inhibiting osteoblast differentiation [18]. In addition, Col X is a marker of chondrocyte differentiation and maturation. PTHrP selectively inhibits Col X gene expression and protein synthesis, so as to inhibit chondrocyte differentiation and maturation [19,20].
Ihh is known to be the most important cytokine to regulate chondrocyte proliferation and differentiation, playing a vital role during the cartilaginous osteogenesis [21,22]. Ihh was found to be the primary upstream molecule of PTHrP, using the Ihh −/− mice. In the absence of hedgehog protein, Ihh inhibits the activity of transmembrane protein Smoothened (Smo). With low levels of Ihh synthesis, hypertrophic chondrocytes at the growth plate expanded and chondrocyte proliferation decreased, resulting in dwarf malformation of the short limb. In the Ihh −/− model, impaired chondrocyte proliferation and differentiation, or impaired osteoblast formation and mineralized bone structures, were noted, indicating that Ihh participates in regulating the cartilaginous osteogenesis [15,16]. Besides, multiple bone defect diseases are related to Ihh gene mutation.
It was reported that when PTHrP was added, PTHrP and Ihh gene expression levels decreased significantly over time. Therefore, excess PTHrP inhibited Ihh expression and synthesis, resulting in decreased PTHrP expression. A study with the Ihh/PTHrP signaling pathway model proved that activation of Ihh signaling upregulates PTHrP expression. PTHrP increases the number of proliferating chondrocytes by inhibiting chondrocyte hypertrophy. At the same time, Ihh and PTHrP precisely regulate the growth of long bone through a feedback regulatory mechanism [23,24].
The Ihh/PTHrP signaling pathway is as follows: hedgehog protein combines with Ihh receptor patched (Ptch) to stimulate a series of complex regulations. Transmembrane protein Smoothened (Smo) is activated, which directly induces transcriptional regulation of its target gene Ihh receptors [25], leading to increased PTHrP synthesis in chondrocytes and elevated PTHrP level around the chondrocytes, diffusing to the anterior hypertrophic chondrocyte region, promoting chondrocyte proliferation, and inhibiting chondrocyte differentiation. Van Donkelaar and Huiskes [9] proved that PTHrP-related factors are major regulatory molecules for chondrocyte hypertrophy, and Ihh-related factors are major regulatory molecules for chondrocyte proliferation. Ihh and PTHrP play different roles during chondrocyte proliferation and differentiation. Ihh regulates chondrocyte proliferation and differentiation during the resting stage through induction of its downstream signaling molecule PTHrP, thus maintaining the properties and functions of chondrocytes [26].
Ihh signaling pathway inhibitor cyclopamine is a plant steroidal alkaloid that inhibits Ihh signaling through antagonism with Smo [27,28]. Some studies also indicate that cyclopamine may inhibit gene expression of Ihh pathwayrelated signaling molecules including PTHrP, Ihh, and Ptch [29]. The downregulation of target genes is dose dependent. Therefore, cyclopamine inhibits Ihh signaling pathway effectively. According to studies by Mak et al. [30], Shimoyama et al. [31], and Mak et al. [32], 10 M cyclopamine, which is the half-effective inhibition concentration, evidently deferred chondrocyte hypertrophy and proliferation, indicating that PTHrP might induce fibrochondrocyte proliferation and inhibit its maturation. In this experiment, cyclopamine blocked PTHrP upstream signaling molecule Ihh and decreased PTHrP gene expression over time. Therefore, this study proved that PTHrP expression is affected by its upstream signaling molecule Ihh.
Fibrochondrocytes specifically synthesize Col I and Col II. It was shown that Col I and Col II were replaced by Col X along with the differentiation of chondrocyte toward hypertrophic fibrous chondrocytes, and acidic glycosaminoglycan secretion decreased within the mesochondrium. Meanwhile, cell activities decreased. Therefore, Col X was considered to be an important parameter for assessing fibrochondrocyte proliferation and differentiation [33]. Col X is used to evaluate the degree of fibrochondrocyte differentiation. The results showed that PTHrP evidently promoted Col I and Col II gene expression and protein synthesis and inhibited Col X gene expression and protein synthesis, indicating that PTHrP may induce fibrochondrocyte proliferation and inhibit its maturation. When its upstream Ihh signaling pathway was blocked by cyclopamine, PTHrP gene expression and protein synthesis both decreased. In contrast, Col X gene expression and protein synthesis both increased, indicating that Ihh promotes fibrochondrocyte maturation and hypertrophy in entheses. In the Ihh/PTHrP double-intervention group, Col I and Col II gene expression and protein synthesis increased. Therefore, PTHrP might induce Col I and Col II gene expression and protein synthesis to promote chondrocyte proliferation. This effect was not affected by Ihh signal block. Ihh induced PTHrP expression, and PTHrP overexpression downregulated Ihh. PTHrP and Ihh coordinately regulated chondrocyte proliferation and differentiation in entheses through a negative feedback mechanism. This proved that Ihh signal regulates fibrochondrocyte differentiation in entheses through PTHrP.
In summary, Ihh/PTHrP signaling pathway is the crucial regulator in regulating fibrochondrocyte development in entheses. Ihh is synthesized by anterior hypertrophic chondrocytes of the fibrous cartilaginous layer in entheses. By autocrine and paracrine secretion, Ihh combines with transmembrane Ihh receptors on fibrochondrocytes during the G0/G1 stage within the surface layer of fibrous cartilaginous layer to regulate and induce fibrochondrocyte proliferation and differentiation and increase PTHrP synthesis. PTHrP inhibits hypertrophic differentiation of chondrocytes within the fibrous cartilaginous zone of entheses. Meanwhile, PTHrP inhibits Ihh synthesis in anterior hypertrophic chondrocytes in the fibrous cartilaginous layer.
Ihh/PTHrP signaling pathway is an important regulator for fibrochondrocyte proliferation and differentiation in entheses. Ihh signal controls fibrochondrocyte differentiation in entheses through PTHrP to promote fibrochondrocyte hypertrophy and maturation, while PTHrP induces fibrochondrocyte proliferation but inhibits the differentiation and maturation. Ihh and PTHrP signals coexist in entheses, forming a signaling pathway to interactively regulate fibrochondrocyte proliferation and differentiation through a negative feedback loop, ensuring the physiological process of entochondrostosis, maintaining the balance of the fibrous cartilaginous layer growth and differentiation and thereby protecting the physiological functions of entheses.
Our study has limitations. Despite the fact that FX-4000 system can simulate the investigating cellular responses to mechanical loads with a maximum ability, it is still unable to simulate the mechanical loads of the Entheses of pig Achilles tendon during different motion.

Conclusions
In this study, Ihh and PTHrP coregulate fibrochondrocyte proliferation and differentiation in entheses through negative feedback regulation. Low tensile strength of CTS (4%, 3 h, 1 Hz) promotes cell proliferation, and high tensile strength of CTS (12%, 12 h, 1 Hz) causes differentiation of fibrochondrocytes. This phenomenon is regulated by stress stimulation through the Ihh/PTHrP signaling pathway.