Dysfunction of Collagen Synthesis and Secretion in Chondrocytes Induced by Wisp3 Mutation

Wisp3 gene mutation was shown to cause spondyloepiphyseal dysplasia tarda with progressive arthropathy (SRDT-PA), but the underlying mechanism is not clear. To clarify this mechanism, we constructed the wild and mutated Wisp3 expression vectors and transfected into human chondrocytes lines C-20/A4; Wisp3 proteins subcellular localization, cell proliferation, cell apoptosis, and Wisp3-mediated gene expression were determined, and dynamic secretion of collagen in transfected chondrocytes was analyzed by 14C-proline incorporation experiment. Mutated Wisp3 protein increased proliferation activity, decreased apoptosis of C-20/A4 cells, and aggregated abnormally in cytoplasm. Expression of collagen II was also downregulated in C-20/A4 cells transfected with mutated Wisp3. Wild type Wisp3 transfection increased intracellular collagen content and extracellular collagen secretion, but the mutated Wisp3 lost this function, and the peak phase of collagen secretion was delayed in mutated Wisp3 transfected cells. Thus abnormal protein distribution, cell proliferation, collagen synthesis, and secretion in Wisp3 mutated chondrocytes might contribute to the pathogenesis of SEDT-PA.


Introduction
Wnt-1-induced secreted protein 3 (Wisp3/CCN6) is a cysteine-rich protein that belongs to the cysteine-rich 61connective tissue growth factor, nephroblastoma overexpressed CCN family members, maps to chromosome 6q21-22, and encodes a 354 amino acid secreted protein [1]. Wisp3 proteins are characterized by an N-terminal secretory signal followed by four structural domains with partial sequence identity to insulin-like growth factor binding protein (IGFBP) (GCGCCXXC); Von Willebrand factor type C like motif, thrombospondin type 1 module, and a Cterminal cysteine knot-like domain (CK) putatively involved in dimerization [1,2], and IGFBP can be upregulated by implementation of exercise [3]. The members of CCN family are multifunctional in which they are involved in regulation of cell adhesion, migration, proliferation, growth arrest, survival, apoptosis, differentiation, endochondral ossification, and extracellular matrix production [4][5][6].
Wisp3 mutations have been demonstrated in most patients of an autosomal recessive hereditary cartilage metabolic disorder, spondyloepiphyseal dysplasia tarda with progressive arthropathy (SEDT-PA), or progressive pseudorheumatoid dysplasia (PPD), which characterized by deformation and limitation of most large and small joints clinically, and continuous degeneration and loss of articular cartilage pathologically [7][8][9][10][11]. In our previous work, we found a novel compound mutation (840delT/T1000C) of Wisp3 in Chinese PPD kindred [12,13]; the two probands carried a substitution mutation (1000T → C, Ser334Pro) in paternal allele, and a deletion (840delT) mutation in maternal allele that caused a truncated Wisp3 protein to miss 43 residues in C-terminus [14], and we also discovered the biological behavior changes of the articular chondrocytes (ACs) separated from the patients [15]. Wisp3 also had growth-, invasion-, and angiogenesis-inhibitory functions in inflammatory breast cancer (IBC) in vitro and in vivo [16] and was a key genetic determinant of the IBC phenotype [17]. However, the precise action of Wisp3 in cartilage maintenance and metabolism and the mechanisms of SEDT-PA/PPD caused by Wisp3 mutations have not been elucidated.
The present study was undertaken to investigate the subcellular localization and function of mutant Wisp3 in chondrocytes. The results suggest that mutated Wisp3 protein aggregated abnormally in cytoplasm, and mutated Wisp3 failed to inhibit cell proliferation and modulate the expression of type II collagen in chondrocytes, which may be an important molecule mechanism involved in the pathogenesis of SEDT-PA/PPD.

Cell Cultures.
The immortalized human chondrocytes cell lines C-20/A4 were derived from human juvenile costal cartilage and generated by infection with a replication defective retroviral vector expressing SV40 large T antigen. Cultures of C-20/A4 cells were maintained in DMEM/Ham's F-12(1 : 1,v/v) supplemented with 10% fetal bovine serum (FBS, Invitrogen, Carlsbad, USA) in a 5%CO 2 incubator at 37 ∘ C and passaged at subconfluence every 5-6 days.

Cell Transfection.
Lipofectamine was used for transfecting C-20/A4 chondrocytes cell lines with the recombined plasmids and empty vector constructs. Briefly, cells (2.5∼5 × 10 5 /mL) were plated 1 day before transfection in 6-well tissue culture plates (2 mL/well) and incubated at 37 ∘ C in 5% CO 2 . A complex of the plasmid DNA (<1 g) with 6 L PLUS reagent in 100 L of serum-free, antibiotic-free medium was prepared in a sterile microfuge tube for 15 minutes; dilute 4 L of Lipofectamine into 100 L of serum-free medium and added to each reaction mixture, and incubated at room temperature for additional 15-30 min. A similar complex was prepared for each well of a 6-well plate. The cells in each well of the plate were washed with sterile PBS and then added 800 L serum-free medium and the transfection mixture drop wise to each plate, and incubated for 4 h, after which 1 mL of culture medium with 5% FBS was added to each well. After 24 hours, the transfection mixture was replaced with fresh culture medium containing 10% FBS. The incubation was continued for an additional 24-26 hours, and the cells were used for observation by laser scanning confocal microscopy (LSCM) and harvested for either RNA or protein extraction. After 24 hours of transfection, the cells were placed in 25 cm 2 flasks for stable transfection with selection by G418 (400 L /mL).

Wild and Mutant Wisp3 Proteins Subcellular localization.
After 48 h of transiently transfection with WT-Wisp3/ pEGFP-C2, MUT 1000T/C /pEGFP-C2, MUT 840delT /pEGFP-C2, or empty vector, cells were rinsed once with PBS after removal of culture medium, and then fixed for 15 min with freshly prepared 4% paraformaldehyde, and then incubated with 0.25% Triton X100 at 37 ∘ C for 20 min. 4 -6-Diamino-2-phenylindole-2HCl (DAPI; Sigma) was used at a final concentration of 100 ng/mL to stain cell nuclei. After washing three times with PBS at room temperature for 10 min, the fluorescence was observed under LSCM.

Cell Cycle and Apoptosis
Analysis. Cells stably transfected with MUT 1000T/C /pcDNA3.1(+), MUT 840delT /pcDNA3.1(+), or empty vector were seeded into in 25 cm 2 flasks at a density of 2 × 10 5 cells/mL and cultured for 24 h. After cultured in serum-free medium for 24 h, cells were digested with 0.05% trypsin-EDTA, rinsed with PBS, fixed with 75% ethanol over night at 4 ∘ C, and stained with propidium iodide. Cell cycle and apoptosis were evaluated using FAC flow cytometry (BD Biosciences). Cell proliferation index (PI) was calculated using the equation (PI) = (G2+S)/(G1+S+G2). Apoptosis was also studied morphologically using fluorescent dyes that intercalate DNA. Acridine orange stains DNA bright green, allowing visualization of the nuclear chromatin pattern. Apoptotic cells have condensed chromatin that is uniformly stained. Ethidium bromide stains DNA orange, but is excluded by viable cells. Cells stably transfected with MUT 1000T/C /pcDNA3.1(+), MUT 840delT /pcDNA3.1(+), or empty vector were stained by acridine orange and ethidium bromide, respectively, and observed using LSCM.

RNA Extraction and RT-PCR.
The C-20/A4 chondrocytes were transfected with WT-Wisp3, MUT 1000T/C , MUT 840delT , or empty vector, and the total RNA was extracted using TRizol Reagent (Invitrogen). RNA was treated with RNase-free DNase (Promega). Reverse transcriptase-PCR (RT-PCR) was performed using a reverse transcription kit according to the instructions of the manufacturer (Invitrogen). Primers specific for type II collagen, type I collagen, SOX9, fibronectin, MMP-1, and -actin were used for estimating the levels of expression of the corresponding mRNA. During cDNA synthesis, 2 g of RNA was used for each specimen, and 30 cycles of PCR were carried out. The -actin gene was used as an internal control. Table 1 summarizes the primer pairs and experimental conditions used for RT-PCR analysis.

Preparation of Whole Cell Protein Lysates and Western
Blot. To prepare whole cell lysates, cells transfected with WT-Wisp3, MUT 1000T/C , MUT 840delT , or empty vector were rinsed once with precooling PBS after removal of culture medium and treated with precooling Triton lyses buffer (50 mmol/L Tris-HCL, PH8.0 containing 150 mmol/L NaCL, 1%Triton X100, 10 mmol/L EDTA, 0.2%NaN 3 , 10 g/mL Aprotinin, and 1 g/mL phenylmethylsulfonyl fluoride) on ice for 20 minutes and the protein concentrations were determined using Bradford protein assay. Approximately 50 g of each cell lysate was mixed with 2×SDS gel-loading buffer (100 mmol/L Tris-HCL, PH6.8, and 200 mmol/L DDT, 4% SDS, 0.2% bromphenol blue, and 20% glycerol) and then heated to 95 ∘ C for 5 min. The samples were loaded onto a polyacrylamide gel (7.5 for type II collagen, 12% for Wisp3 and -actin), and prestained molecular weight standard (Bio-Rad, CA, USA) was also loaded onto the gels. After electrophoresis, the SDS-PAGE separated proteins were transferred to a nitrocellulose (GE, Pittsburgh, USA). The membrane was blocked with 5% nonfat milk in PBS for 60 min and then incubated with 2 g/mL goat monoclonal antihuman type II collagen, Wisp3, or -actin (Santa Cruz, CA, USA) for 60 min. After extensive washing with PBS, the membrane was reprobed with mouse anti-goat IgG conjugated with horseradish peroxidase (GE) at 1 : 1000 in PBS for 1 h at room temperature. Blots were visualized with chemiluminescence as described previously [18].
2.9. 14 C-Proline Incorporation Analysis. Chondrocytes were seeded into 24-well plates (5 × 10 4 cells/cm 2 ) in DMEM with 10% FBS for 24 hours, then cultured in 500 L of serum-free DMEM for 4 hours. For 14 C-proline incorporation, each well was added with 10 Ci of 14 C-proline (100 Ci/mL) (GE) and 100 g/mL aminopropionitrile, then incubated for 2 h; cells were rinsed five times with PBS, and complete medium was added and incubated at 37 ∘ C for 0, 30, 60, 120, 180, 240, and 300 minutes; supernatant and cell lysates were collected at these time points. For the collection of cell lysates, cells were rinsed two times with PBS after the supernatant collection, two times with 5% cold trichloroacetic acid, two times with 80% ethanol and finally lysed at 37 ∘ C for 2 h in 0.5 mL of 10 mM EDTA; 0.2 mL aliquots of the lysates and supernatant were dissolved in 10 mL Ecoscint H (Prolabo, Briare, France) separately and counted by scintillation; the quantification of intracellular collagen content and extracellular secretion was determined by the radioactivity in the cell lysate and supernatant, and the ratio of extracellular collagen secretion to intracellular collagen content was counted.

Statistical
Analysis. SPSS 11.0 software was used for statistical analysis and data are presented as the mean ± SD, with the exception of gene analysis data. Data were compared using one-way ANOVA or the student's -test. All experiments were repeated at least 3 times; the representative experiments are shown.
Overproliferation of cells stably transfected with mutant wisp3 was further demonstrated by flow cytometry analysis, which indicated that the cell numbers in the G2-M plus S phases were significantly higher than that of control cells (33.6±4.0%, < 0.05, Figure 2(b)), with a proliferation index of 49.8 ± 5.0% and 53.2 ± 4.5%, respectively (Figures 2(c) and 2(d)).

Dysfunction of Collagen Production in Cells
In contrast, minimal changes were observed in the levels of mRNA of type I collagen, SOX9, and fibronectin in response to either wild or mutant Wisp3 (Figures 4(c), 4(d), and 4(e)). The mRNA expression of MMP-1, which had been found dramatically decreased in articular chondrocytes separated from SEDT-PA/PPD patient [15] (Figure 4(f)), wasn't changed in the mutant Wisp3 transfected chondrocytes.

Abnormal Intracellular Collagen Content and Secretion in Mutant
Chondrocyte. By 14 C-proline incorporation analysis, very low radioactivity was detected in the supernatant of and cell lysate of C-20/A4 cells transfected with control vectors, which indicated that very low collagen synthesis and secretion in this cell line (Figures 6(a) and 6(b)), and the ratio of extracellular collagen secretion to intracellular content is approximately 1. However, in wild type Wisp3 stably transfected C-20/A4 cells, high radioactivity, were detected in the culture supernatant (3000 CPM to 7000 CPM) and cell lysate (700 CPM to 1000 CPM); the peak collagen secretion and intracellular content were appeared at 120 min and 60 min separately after refreshment of the complete medium, compared to C-20/A4 cells transfected with control vector; Wisp3 increased the intracellular collagen content to about 5-10 times ( < 0.01), and especially increased the extracellular collagen secretion to 10-20 times ( < 0.01), and the ratio of extracellular collagen secretion to intracellular content is 3.5-10 ( Figure 6(c)). In mutant Wisp3 (MUT 840delT and MUT 1000T/C ) transfected cells, the peak collagen secretion and intracellular content were backward to 120 min and 180 min separately, although the radioactivity of collagen secretion was slightly higher than that of intracellular collagen content, the extracellular collagen secretion was decreased obviously compared to the wild type Wisp3 transfected cells, and the ratio of extracellular collagen secretion to intracellular content is about 1.5.

Discussion
PPD was attributed to mutations of Wisp3 gene; we previously identified a novel compound heterozygous mutation (840delT/T1000C) of Wisp3 in a SEDT-PA/PPD family, and this mutation results in a dramatic decrease in the tensile strength of articular cartilage; however, the detail mechanism is not clear.
By bioinformatics analysis, we predicted that the compound heterozygous mutation formed a truncated Wisp protein and a Ser334Pro mutated proteins [14]. The 3Dconformational change of the 840delT truncated mutant International Journal of Endocrinology Wisp3 protein is the single long peptide loop in the region from signal peptide to the beginning 24 amino acid residues in the first domain (IGFBP) which was subjected to folding into two smaller cross-loops accompanied with a much shorter C-terminus. It has been noted that the function of the first (IGFBP) domain of Wisp3 is involved in inhibiting the function of IGF-1 to the chondrocytes and the fourth (CK) domain is involved in disulfide-linked dimerisation and is necessary for dimer formation in the endoplasmic reticulum, an important function for the establishment and maintenance of normal 3D-conformation of Wisp3 protein [18][19][20]. Through GFP labeled protein localization analysis, we found that wild type Wisp3 protein did localize in cytoplasm and cell membrane of C-20/A4 cells, but the two mutated Wisp3 proteins aggregated abnormally in cytoplasm of C-20/A4 cells transfected with MUT 1000T/C and MUT 840delT . It needs further research to validate the hypothesis that 3Dconformational change causes localization change of mutated Wisp3 protein.
Wisp3 belongs to the CCN family of proteins, which play important roles in development during chondrogenesis and enchondromatosis and encode cysteine-rich secreted proteins with roles in cell growth and differentiation [21]. To investigate the effects of the T1000C and 840delT mutations on Wisp3 function in chondrocytes, we compared biological behaviors in C-20/A4 cells transfected separately with WT-Wisp3, MUT 1000T/C , and MUT 840delT . MUT 1000T/C Wisp3 and MUT 840delT Wisp3 increased proliferation activity as well as decreased apoptosis of C-20/A4 cells obviously, which shared the phenotype of articular chondrocytes (ACs), separated from SEDT-PA patients we described before [15]. Therefore, inhibition of cell proliferation and promotion of precursor cell differentiation are major effects of Wisp3 on chondrocytes, through which Wisp3 modulates the balance of cartilage metabolism.
We previously found that PPD cartilage had lost its flexibility, and the main matrix component of cartilage is collagen, so we detected the effect of Wisp3 gene mutation on the collagen expression in chondrocytes. The results demonstrated that both mutant Wisp3 lose the function to modulate the expression of cartilage specific matrix type II collagen when compared with wild Wisp3, which consisted of results in C-28/I2 and T/C-28a2 cells transfected with another SEDT-PA related Wisp3 mutation (Cis78-Arg) [22], but the modulation effect of Wisp3 may not be via activation of SOX9 in our study since no change of SOX expression was found. After the collagen synthesis, it need to be secreted into the extracellular matrix, if the collagen secretion was changed by gene mutation or 3D-conformational alteration, the function of cartilage will be abnormal, and to further study the dynamic collagen synthesis and secretion, we use 14 C-proline, which is the major material of collagen synthesis and a major determinant of collagen tertiary structure, to label the new synthesized collagen, through detection of the radioactivity in cell lysate and supernatant to quantify the intracellular collagen content and extracellular secretion at different time points. Compared to the wild type Wisp3, MUT 1000T/C or MUT 840delT Wisp3 lost the function of increasing the extracellular collagen secretion, delaying the intracellular collagen synthesis, which is one of the important mechanisms for the collage size and density decrease in PPD cartilage described previously. However, we could not find the difference of MMP-1 mRNA levels between the C-20/A4 cells transfected with wild and mutant Wisp3, which was dramatically decreased in ACs separated from SEDT-PA/PPD patients compared with normal ACs. The paradoxical phenomenon may be related to the following causes: (1) C-20/A4 is an immortalized cell line derived from human juvenile costal cartilage and is highly proliferative and not contact-inhibited compared with primary cells, which may have influence on the expressions of matrix and other genes at reasonable levels [23].

Conclusions
Wisp3 mutations resulted in abnormal protein distribution and dysfunction of cell proliferation, collagen production, and dynamic secretion in chondrocytes, which may be involved in the pathogenesis of SEDT-PA.