Tumor Proteins D52 and D54 Have Opposite Effects on the Terminal Differentiation of Chondrocytes

The tumor protein D (TPD) family consists of four members, TPD52, TPD53, TPD54, and TPD55. The physiological roles of these genes in normal tissues, including epidermal and mesenchymal tissues, have rarely been reported. Herein, we examined the expression of TPD52 and TPD54 genes in cartilage in vivo and in vitro and investigated their involvement in the proliferation and differentiation of chondrocytes in vitro. TPD52 and TPD54 were uniformly expressed in articular cartilage and trabecular bone and were scarcely expressed in the epiphyseal growth plate. In MC3T3E-1 cells, the expressions of TPD52 and TPD54 were increased in a differentiation-dependent manner. In contrast, their expressions were decreased in ATDC5 cells. In ATDC5 cells, overexpression of TPD52 decreased alkaline phosphatase (ALPase) activity, while knock-down of TPD52 showed little effect. In contrast, overexpression of TPD54 enhanced ALPase activity, Ca2+ deposition, and the expressions of type X collagen and ALPase genes, while knock-down of TPD54 reduced them. The results revealed that TPD52 inhibits and that TPD54 promotes the terminal differentiation of a chondrocyte cell line. As such, we report for the first time the important roles of TPD52 and TPD54, which work oppositely, in the terminal differentiation of chondrocytes during endochondral ossification.

The vertebrate skeleton develops via two separate mechanisms: intramembranous ossification and endochondral ossification. The flat bones of the skull, parts of the craniofacial skeleton, and the clavicles are formed by intramembranous ossification, and the rest of the craniofacial bones and the axial and appendicular skeleton are generated by endochondral ossification (reviewed in [25]). In both processes, the first step is condensation of mesenchymal progenitor cells at the site of the future bones. In intramembranous ossification, the condensed cells directly differentiate into bone-producing osteoblasts (reviewed in [26]). In contrast, in endochondral 2 BioMed Research International ossification, a cartilaginous template is later replaced by bone. Cartilage elements grow from the proliferation of chondrocytes with a type II collagen-rich extracellular matrix (ECM); these chondrocytes differentiate into prehypertrophic chondrocytes that produce increased levels of type X collagen and alkaline phosphatase (ALPase) when they finally undergo apoptosis [25]. Every step of endochondral ossification is regulated by the concerted actions of various growth factors, signaling molecules, and cytokines, such as type I, II, and X collagens, ECM components (e.g., osteonectin, osteocalcin, osteopontin, fibronectin, and CD44), and early response genes, such as c-fos [27].
In the present study, we focused on the tissue-specific distribution of the TPD52 and TPD54 in bone and cartilage tissue in vivo and the cell-specific physiological roles of those proteins on proliferation and differentiation of chondrocytes in endochondral ossification in vitro. As a result, we found that TPD52 and TPD54 regulate the hypertrophy of chondrocytes in opposite ways and that they might play important roles in the terminal differentiation of chondrocytes in cartilage tissue during endochondral ossification.

2.2.
Immunohistochemistry. The sacrifice of the experimental animal complied with the Showa University Guidelines for Animal Experiments, and the experimental protocol was approved by the Animal Experimentation Committee of Showa University (approval number 28D009). A 12-week-old Slc:SD rat (Sankyo Labo Service, Tokyo, Japan) was sacrificed, and the tibias were removed and fixed in 10% buffered neutral formalin (Wako). The fixed specimens were decalcified with Kalkitox (Wako) and embedded in paraffin. Then, the sections were cut on a rotary microtome, were mounted on microscope slides, and were subjected to Hematoxylin-Eosin (H-E) staining as described previously [28]. Thereafter, the sections were incubated overnight at 4 ∘ C in a humid chamber with primary antibodies: anti-TPD52 antibody (1/100 dilution; Santa Cruz, Dallas, TX, USA) or anti-TPD54 antibody (1/200 dilution; Proteintech, Rosemont, IL, USA). On the next day, sections were incubated with the secondary antibody (EnVision+ System-HRP Labelled Polymer Anti-Rabbit; Dako, Carpinteria, CA, USA). Sections were reacted with the Dako Liquid DAB+ Substrate Chromogen System (Dako) and examined under a microscope, and photographs were taken.

RNA Purification and RT-qPCR.
The cells were seeded at a density of 10,000 cells/well in a 12-well tissue culture plate and cultured for 7 days. Total cellular RNA was purified using TRIzol (Life Technologies) according to the manufacturer's protocol. Ten nanograms of total RNA was reverse-transcribed using a commercial kit (iScript cDNA Synthesis Kit; Bio-Rad), and an aliquot of the reaction mixture (1/20) was used for the subsequent qPCR reaction. qPCR was carried out with a KAPA SYBR FAST qPCR kit (Kapa Biosystems, Boston, MA, USA), and statistical analysis was performed using Bio-Rad iQ5 analysis software (Bio-Rad). The fold changes in gene expression were calculated using the 2 −ΔΔCt method. The gene expression levels were first normalized to GAPDH within each sample group. All of the primer sequences for RT-qPCR are shown in Table 1.

Molecular Constructs, Small Interfering RNAs (siRNAs)
, and Transfection. The coding regions of mouse TPD52 and TPD54 cDNAs were amplified by an RT-PCR technique using single-stranded cDNA reverse-transcribed from total RNA of ATDC5 cells as a template. The sequences of the primer pairs for the coding regions of mouse TPD52 and TPD54 are shown in Table 2. The sense and antisense primers harbor Bgl I and Kpn I sites, respectively, and the amplicons were double-digested with the enzymes and were inserted into the corresponding site of pCMV-HA (Clontech, Mountain View, CA, USA) as described previously [24]. Proper constructs were confirmed by nucleotide sequencing using an ABI PRISM 310 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) and a BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). Control siRNA and siRNAs for mouse TPD52 and TPD54 genes were purchased from Sigma-Aldrich. The vectors and siRNAs were transfected into cells in a 6-well tissue culture plate using Lipofectamine 2000 (Life Technologies) according to the manufacturer's protocol.
2.6. Biochemical Assays. The transfected cells were reseeded at a density of 1,000 cells/well in a 48-well tissue culture plate and cultured for 7 days. Thereafter, the cells were lysed with 0.3 ml of 0.02% Triton X-100 (Sigma-Aldrich) in a physiological saline. DNA content, ALPase activity, sulfated glycosaminoglycan (GAG), and calcium (Ca 2+ ) deposition were measured as described previously [30,31].

Cytohistochemistry.
Cells were seeded at a density of 1,000 cells/well in 48-well tissue culture plates and allowed to grow to maturation for 7 days. Then, the cells were stained for ALPase activity by crystal violet and toluidine blue staining as described previously [30,31].

Statistical Analysis.
Unless otherwise specified, all experiments were repeated at least three times, and similar results were obtained in the repeated experiments. Statistical analysis of the repeatability of the assay results was carried out using an unpaired Student's t-test. Data are expressed as means ± standard deviation of triplicate data. Significance was determined at * < 0.05.

Expression of TPD52 and TPD54 Proteins in Bone and
Cartilage In Vivo and In Vitro. First, we examined the expressions of TPD52 and TPD54 in the proximal tibial epiphysis of a 12week-old rat by immunohistochemical staining (Figure 1(a)). TPD52 and TPD54 were uniformly expressed in articular cartilage and trabecular bone but were scarcely expressed in the epiphyseal plate. Based on these in vivo results, the expressions of TPD52 and TPD54 proteins and genes were examined in three cell lines, that is, MC3T3-E1, ATDC5, and RAW264.7 with or without differentiation-inducing supplementation, by western blot analysis (Figure 1(b)) and RT-qPCR (Figure 1(c)). In MC3T3-E1 cells, the expression of the TPD52 gene was increased by an osteogenic stimulation, whereas the expressions of the TPD54 gene and protein were decreased by that stimulation. In contrast, in ATDC5 cells, mRNA levels of TPD52 and TPD54 were decreased by the differentiation-inducing stimulation. In RAW264.7 cells, TPD52 and TPD54 were uniformly expressed regardless of the osteoclast-inducing stimulation. These results showed that TPD52 family proteins might be involved in the proliferation and/or differentiation of osteoblasts and chondrocytes, but not of osteoclasts.

TPD52 and TPD54 Have Little Involvement in the Proliferation and Differentiation of Osteoblasts.
Since the results of the previous subsection led to a hypothesis that the TPD52 and TPD54 genes might play an important role in the proliferation and/or differentiation of osteoblasts and chondrocytes, we initially investigated these effects in MC3T3E1 cells by inducing the overexpression or knockdown of TPD52 and TPD54 using cytomegalovirus-(CMV-) promoter-driven-hemagglutinin-(HA-) tagged expression vectors and small interfering RNAs (siRNAs), respectively, followed by cell-biological assays and RT-qPCR ( Figure 2). Neither overexpression nor knock-down of the genes affected cell proliferation (Figures 2(a) and 2(f)). The overexpression (Figures 2(b) to 2(e)) of TPD52 and TPD54 showed little effect ( > 0.05) on the differentiation of the cells. Knockdown of TPD54 induced mRNA expression of ALPase significantly (Figure 2(i)), while the increasing effect on ALPase activity was little (Figure 2(g)). Also, knock-down of TPD52 decreased mRNA expression of ALPase (Figure 2(i)). The opposite results were observed for Ca 2+ deposition and osteocalcin mRNA expression (Figures 2(h) and 2(j)). These results led us to conclude that TPD52 and TPD54 genes might have little involvement in the proliferation and differentiation of osteoblasts, although more detailed experiments remained.

TPD52 and TPD54 Play More Important Roles in the Terminal Differentiation of Chondrocytes Than in Prehypertrophic
Maturation. Next, we investigated the effects of TPD52 and TPD54 genes on the proliferation and differentiation of ATDC5 cells. Figure 3 shows the successful transfection of HA-tagged TPD52 and TPD54, and of TPD52 and TPD54 siRNAs, resulting in both the overexpression and knockdown of these genes. We focused on roles of those proteins on proliferation and differentiation of chondrocytes in endochondral ossification and investigated the effects of TPD52 and TPD54 genes on the proliferation and differentiation of ATDC5 cells. We investigated the biological effects of     the overexpression (Figure 4) and knock-down ( Figure 5) of these genes in ATDC5 cells. Neither the overexpression nor the knock-down of these genes showed an effect on cell proliferation (Figures 4(a) and 5(a)). The overexpression of TPD52 decreased ALPase activity (Figure 4(c)), whereas the overexpression of TPD54 enhanced ALPase activity and its gene expression (Figures 4(c) and 4(g)), as well as Ca2+ deposition (Figure 4(d)) and type X collagen gene expression (Figure 4(h)). On the other hand, knock-down of TPD52 showed less effects ( Figures 5(b) to 5(h)), and of note, knock-down of TPD54 significantly reduced ALPase activity and its gene expression (Figures 5(c) and 5(g)), as well as type X collagen gene expression ( Figure 5(h)). However, the maturation markers of prehypertrophic chondrocytes (i.e., type II collagen and aggrecan) were barely altered by the overexpression or knock-down of the two genes ( Figures  4(b), 4(e), 4(f), 5(b), 5(e), and 5(f)). Similar results were observed in cytohistochemistry ( Figure 6). The overexpression of TPD52 decreased ALPase activity, and the knockdown of TPD54 did not show any modulating effect on that decrease. On the other hand, the overexpression of TPD54 increased ALPase activity. In addition, only small effects were seen on toluidine blue or crystal violet staining.

Discussion
TPD55 was found to be limited to normal testis [7]. On the other hand, the other members (TPD52, TPD53, and TPD54) have been reported to be highly expressed in various cancers. We recently reported that TPD52 and TPD54 have opposite effect in oral squamous cell carcinoma-derived cell lines [23,24]. However, the expressional distributions and physiological roles of TPD52 family genes in normal tissues, including epidermal and mesenchymal ones, are not well understood. Moreover, there are no reports on roles of these proteins in endochondral ossification. Therefore, in the present study, we sought to identify the physiological roles of TPD52 and TPD54 proteins in cartilage metabolism and revealed for the first time in the world that TPD52 and TPD54 are proteins that are involved in hypertrophy. Ummanni et al. [32] reported that TPD52 expression activates Akt/phosphoinositide-3 kinase (PI3K) signaling, and we recently [24] demonstrated that TPD54 is a negative regulator of Akt/PI3K signaling and decreases the migration and adhesion of oral squamous cell carcinoma cells. In chondrocytes, Akt/PI3K signaling is required for physiological hypertrophic cell differentiation and endochondral bone growth [33], whereas Kita et al. [34] reported that the Akt/PI3K pathway blocks the terminal differentiation of chondrocytes. In addition, p21 cip1 and p27 waf1 , which are located downstream of the Akt/PI3K pathway (reviewed in [35]), are negative regulators of the proliferative zone in chondrogenesis and lead to apoptosis in preparation for ossification [36]. The TPD52 TPD54   TPD52 TPD54   TPD52 TPD54   TPD52 TPD54   TPD52 TPD54   TPD52 TPD54   TPD52 TPD54   TPD52 TPD54 Biochemical assays RT-qPCR leads to the hypothesis that TPD52 might enhance cell proliferation through the Akt/PI3K signaling pathway and maintain the prehypertrophic state of chondrocytes. On the other hand, TPD54 might terminate the prehypertrophic state of chondrocytes and initiate the start of terminal differentiation. As such, the expressions of TPD52 and TPD54 might concordantly play an important role in the regulation of the terminal differentiation of chondrocytes. It is also suggested that some kind of cartilaginous abnormality might occur if the balance between TPD52 and TPD54 expressions collapses. Taken together, the results from the present study showed that TPD52 and TPD54 play an important role in terminal differentiation of chondrocytes with opposite effects on the terminal differentiation during endochondral ossification. In order to address the points, more detailed investigations using the knock-out mouse are ongoing; we will be able to report in the future.

Conclusions
The present study revealed that overexpression of TPD52 inhibits the terminal differentiation phenotypes for prehypertrophic chondrocyte, whereas overexpression of TPD54 promotes them. However, overexpressions of those genes showed little effects on proliferation and maturating phenotypes for proliferating chondrocytes. Consequently, we report for the first time the important roles of TPD52 and TPD54, which work oppositely, in the terminal differentiation of chondrocytes during endochondral ossification.

Disclosure
An earlier version of this work was presented as a poster presentation at International Journal of Oral and Maxillofacial Surgery Volume 44, Supplement 1, October 2015, Page e230.

Conflicts of Interest
The authors declare that they have no conflicts of interest.