Molecular cloning, expression analysis, and function of decorin in goat ovarian granulosa cells

Domestic Animal Endocrinology 57 (2016) 108–116

Contents lists available at ScienceDirect

Domestic Animal Endocrinology journal homepage: www.domesticanimalendo.com

Molecular cloning, expression analysis, and function of decorin in goat ovarian granulosa cells J.Y. Peng a, K.X. Gao a, H.Y. Xin a, P. Han a, G.Q. Zhu a, b, B.Y. Cao a, * a b

College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi, P.R. China, 712100 Department of Animal Engineering, Xuzhou Bioengineering Technical College, Xuzhou, Jiangsu, P.R. China, 221006

a r t i c l e i n f o

a b s t r a c t

Article history: Received 21 February 2016 Received in revised form 11 May 2016 Accepted 18 May 2016

Decorin (DCN), a component of the extracellular matrix (ECM), participates in ECM assembly and influences cell proliferation and apoptosis in many mammalian tissues and cells. However, expression and function of DCN in the ovary remain unclear. This study cloned the full-length cDNA of goat DCN obtained from the ovary of an adult goat. Sequence analysis revealed that the putative DCN protein shared a highly conserved amino acid sequence with known mammalian homologs. The tissue distribution of DCN mRNA expression was evaluated by real-time PCR, and the results showed that DCN was widely expressed in the tissues of adult goat. Immunohistochemistry results suggested that DCN protein existed in the granulosa cells and oocytes from all types of follicles and theca cells of antral follicles. Moreover, hCG-induced DCN mRNA expression was significantly reduced by the inhibitors of protein kinase A, PI3K, or p38 kinase (P < 0.05), which are key mediators involved in hCG-induced DCN expression. Overexpression of DCN significantly increased apoptosis and blocked cell cycle progression in cultured granulosa cells (P < 0.05). Western blot analysis also showed that overexpression of DCN upregulated the expression levels of p21 protein (P < 0.05), whereas no effects were observed on the expression of Bax and Bcl-2 and on Bcl-2/Bax ratio (P > 0.05). These findings suggested that DCN regulates the apoptosis and cell cycle of granulosa cells. Ó 2016 Elsevier Inc. All rights reserved.

Keywords: Apoptosis Decorin Goat Granulosa cells hCG

1. Introduction The extracellular matrix (ECM) is a major component of the cellular microenvironment and provides support to cells [1]. Decorin (DCN), a member of the small leucine-rich proteoglycan (SLRP) gene family, is a component of the ECM and plays a crucial role in a variety of physiological processes [2]. DCN also regulates collagen fibril formation and controls cell proliferation [3]. DCN protein is highly conserved and shares structural homologies, such as cysteine residues, leucine-rich repeats (LRRs), and at least one glycosaminoglycan side chain, with those of other species [4]. In addition, DCN is widely expressed in

* Corresponding author. Tel.: þ86 29 87092120; fax: þ86 29 87092164. E-mail address: [email protected] (B.Y. Cao). 0739-7240/$ – see front matter Ó 2016 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.domaniend.2016.05.006

mammalian tissues and can directly regulate multiple growth factors, such as transforming growth factor-b1 [5,6]. DCN also antagonizes a number of receptor tyrosine kinase (RTKs), including epidermal growth factor receptor (EGFR) [7], insulin-like growth factor 1 receptor (IGF-IR) [8], and receptor for hepatocyte growth factor [9]. DCN is expressed in reproductive tissues, including ovary and uterus. In the uterus, high levels of DCN are detected, and the steroids upregulate DCN expression levels [10,11]. In the ovary, DCN is present in normal and tumorous tissues [12–14]. Moreover, overexpression of DCN in ovary cells suppresses cell proliferation [15]. A recent study has also shown that DCN is produced in follicular cells, and treatment with DCN causes rapid phosphorylation of EGFR in ovarian granulosa cells [16]. However, the roles of the DCN in female fertility and ovarian function remain largely unknown.

J.Y. Peng et al. / Domestic Animal Endocrinology 57 (2016) 108–116

Our previous studies have shown that DCN expression is upregulated in ovarian tissues of polytocous goats compared with that in monotocous goats (unpublished data), and hCG induces a rapid and transient DCN expression in cultured granulosa cells of goat [17]. These results suggested that DCN regulates the reproductive function of goats. In this follow-up study, we cloned and characterized the goat DCN gene and investigated its expression in various tissues and in follicles of goat. We also determined the regulatory mechanism of hCG toward DCN expression in goat ovarian granulosa cells and the potential function of DCN in the ovary. 2. Materials and methods 2.1. Reagents hCG was purchased form Sigma-Aldrich (St. Louis, MO, USA). Chemicals and reagents, including H89, LY294002, and Forskolin (FSK), were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Phorbol 12-myristate 13-acetate (PMA) and SB2035850 were purchased from Beyotime Biotechnology (Jiangsu, China). DMEM/F12 and fetal bovine serum were obtained from Life Technologies Inc (Carlsbad, CA, USA). 2.2. Tissue collection and cell culture Healthy adult Guanzhong dairy goats (n ¼ 4, 1–3 years old, cyclic, and in good body condition) were stunned using a captive bolt and then slaughtered to collect different tissues. Eleven tissues, including the uterus, spleen, kidney, heart, liver, lung, ovary, muscle, fat, mammary gland, and oviduct, were immediately collected, cut into small pieces of approximately 1 g in 0.1% w/v diethylpyrocarbonate in water and then immediately placed in liquid nitrogen. All animal procedures presented in this article were approved by the animal ethics committee of the Northwest A&F University (Yangling, China). Goat granulosa cells were collected from the ovaries of Guanzhong dairy goat by using the follicle isolation method as previously described [17]. Briefly, the tissue was first washed with 75% alcohol for 1 min and then washed 3 times with PBS to eliminate alcohol. Small antral follicles (1– 3 mm) were then harvested by an aseptic needle under the stereomicroscope. After washing 3 times with DMEM/F12 medium (Gibco, Grand Island, NY, USA), the small antral follicles were cut into pieces, and then an aseptic needle was used to release the granulosa cells. The COCs and ovarian tissues were discarded under the stereomicroscope. Granulosa cells were harvested by centrifuging (800  g) for 10 min and washing twice in DMEM/F12 medium. The granulosa cells were counted in a hemocytometer, the viability was determined by trypan blue exclusion, and the cells were seeded in 12-well culture plates at a density of 2  105/well in 1 mL of DMEM/F12 with 10% FBS, 100 IU/mL penicillin, and 50 mg/mL streptomycin. The cells were cultured at 37 C in a 5% CO2 atmosphere. After 24 h, the cells were washed twice with PBS and changed with fresh medium (DMEM/F12 with 10-mM HEPES, 20-mM L-glutamine, 100 IU/mL penicillin, 50 mg/mL streptomycin, 0.2% BSA,

109

10 mg/mL transferrin, 4 ng/mL sodium selenite, and 10 ng/mL insulin) for 12 h. The cells were then treated with specific reagents at a time interval indicated in the text. When the reagents were dissolved in dimethylsulfoxide (DMSO), the same DMSO concentration was added into the medium of the control cells. The final concentration of DMSO in cultures was less than 0.05%. At the end of each culture period, the cells were collected for total RNA isolation. 2.3. Cloning, sequence alignment, and phylogenetic analyses of goat DCN The goat DCN was cloned through RT-PCR using the cDNA obtained from the adult ovary of a goat. The primers (forward 50 -GGGCTCCAGTGGCAAATC-30 , reverse 50 CCCGCCGTGAGTTACAGA-30 ) were synthesized based on the DCN cDNA sequences of cattle (NM_173906) and sheep (GAAI01007035). The amplified fragments were visualized in a 1.5% agarose gel, and the products exhibiting the expected size were cut and purified using a TIANgel Midi Purification Kit (Tiangen Bio-tech, Beijing, China). The purified PCR products were ligated into a pMD19-T vector and then transformed into DH5a (Escherichia coli) according to the manufacturer’s instructions. The full-length ORF cDNAs were finally determined through sequencing (Genewiz, Suzhou, China). The deduced amino acid sequences were compared with the sequences in the GenBank database using Basic Local Alignment Search Tool (BLAST) program available from the National Center for Biotechnology Information (NCBI) website (http://www.ncbi.nlm.nih.gov). The signal peptide was predicted using SignalP 4.1 (http://www.cbs. dtu.dk/services/SignalP). Multiple sequence alignment was performed using ClustalW (http://www.ebi.ac.uk/ clustalw), and a phylogenetic tree was constructed by MEGA 5.0 using the neighbor-joining method [18]. 2.4. RNA isolation, reverse transcription, and real-time PCR Total RNA was extracted from 11 different tissues (uterus, spleen, kidney, heart, liver, lung, ovary, muscle, fat, mammary gland, and oviduct) and from cells using RNAiso Plus (TaKaRa, Dalian, China) following the manufacturer’s instructions. The concentration and purity of RNA were determined by measuring the optical density (OD) at 260 and 280 nm wavelengths using an Epoch Microplate Spectrophotometer (BioTek Instruments. Inc., USA). The OD260/ 280 ratios were >1.8 and <2.1 for all of the samples. The total RNAs (500 ng) were used to convert the mRNAs into cDNAs by using a PrimeScript RT reagent (TaKaRa, Dalian, China) according to the manufacturer’s instructions. Real-time (RT) PCR was then performed in a 20-mL reaction vol containing 10 mL of SYBR Premix Ex Taq II (TaKaRa, Dalian, China), 1 mL of template cDNA, and 1 mM of primers by using the CFX Connect Real-Time PCR Detection System (Bio-Rad, CA, USA). The thermal cycling conditions were 95 C for 10 min followed by 40 cycles at 94 C for 15 s, 60 C for 30 s, and 72 C for 30 s. The following primers were used for RT PCR: DCN, 50 -TGGATTGAACCAGATGATCGTC-30 (sense) and 50 -GTCAGCAATGCGGATGTAGGA-30 (antisense); and b-actin, 50 -TGACCCA GATCATGTTTGAGA-30 (sense) and

110

J.Y. Peng et al. / Domestic Animal Endocrinology 57 (2016) 108–116

50 -CAAGGTCCAGACGCAGGAT-30 (antisense). b-actin was used for normalization. Each experiment was independently repeated at least thrice, and the fold change in the expression of each gene was analyzed using the 2DDCt method [19]. 2.5. Plasmid construction and cell transfection DCN cDNA was amplified from granulosa cells by using Pfu DNA polymerase (Fermentas, Shanghai, China) and then cloned into pcDNA3.1 (þ) expression vector (Invitrogen, CA, USA) by using a T4 DNA Ligation Kit (Fermentas, Shanghai, China). The following primers were used: 50 -CGGGATCCGAAATCATGAAGGCAGCTATCATC-30 (sense) and 50 CGGAATTCTGGGAGCTACTTGTAGTTTCCAA-30 (antisense). The PCR conditions were as follows: 95 C for 5 min followed by 32 cycles at 94 C for 30 s, 60 C for 30 s, and 72 C for 2 min; and a final extension of 10 min at 72 C. The PCR product was 1,095 bp long, which was then excised with BamHI and EcoRI (TaKaRa, Dalian, China) and then cloned into pcDNA3.1 (þ). The new vector was named pcDNA3.1DCN. The inserted sequences were confirmed by DNA sequencing. Cell transfection was performed using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) as described by the manufacturer’s instructions. Briefly, granulosa cells were plated, grown to 85%–90% confluence without antibiotics, and then transfected with pcDNA3.1 empty vector or pcDNA3.1-DCN for 24 h. 2.6. Immunohistochemistry Immunohistochemistry was performed according to a previously described protocol [17]. Briefly, the ovaries were fixed in 4% paraformaldehyde for 24 h before paraffin embedding. Paraffin sections (5 mm) were attached to microscope slides, heated at 60 C for 1 h, deparaffinized in xylene, and then rehydrated in a graded series of ethanol. For antigen retrieval, these sections were boiled in 10-mM citrate buffer (pH 6.0) in a microwave oven at 600 W for 15 min and then cooled down to room temperature. After washing with PBS, we blocked endogenous peroxidase activity by incubating the sections in 3% hydrogen peroxide for 10 min. To block nonspecific binding, the sections were incubated in a blocking buffer (3% BSA in PBS) for 45 min at room temperature. The sections were then incubated overnight at 4 C with primary, ie, antibody rabbit anti-DCN polyclonal antibody (1:100, Bioss, bs-1695R, Beijing, China) in PBS. After washing with PBS, the sections were incubated with a biotinylated secondary antibody for 1 h (37 C) and with horseradish peroxidase (HRP)–streptavidin for 15 min before visualization with diaminobenzidine. For negative controls, the primary antibodies were replaced with nonspecific rabbit IgG. Digital images were captured using an Olympus BX53 microscope.

isolated granulosa cells (1  105 cells/mL) from goat follicles were seeded into a 6-well plate as previously described and then transfected with pcDNA3.1 empty vector or with pcDNA3.1-DCN. After 24 h, the treated cells were harvested, washed with PBS, and then treated with 0.25% trypsin. The cells were then resuspended in 500 mL 1  Annexinbinding buffer; 5 mL of Annexin V-FITC, and 10 mL of propidium iodide (PI) were subsequently added followed by incubation for 5 min at room temperature in the dark. The cells were then analyzed by flow cytometry using FlowJo software (FACS Calibur, Becton Dickinson, CA, USA). To investigate the effect of DCN on cell cycle distribution, we detected the granulosa cells (1 105 cells/mL) by using a cell cycle kit (MultiSciences Biotech, Hangzhou, China). The granulosa cells were transfected with pcDNA3.1 empty vector or with pcDNA3.1-DCN for 24 h. The treated cells were harvested, washed with PBS, and then fixed in 75% ethanol overnight at 4 C. After centrifugation and removal of ethanol, the cells were washed once with PBS. The cells were then incubated in 1 mL of staining buffer (A) and 10 mL of reagent B for 30 min at room temperature in the dark. The stained cells were analyzed using a flow cytometer, and the data were calculated using ModFit software. 2.8. Western blot analysis The granulosa cells were harvested and rinsed twice with PBS, lyzed in denaturing lysis buffer–containing protease inhibitors (RIPA, Applygen Technologies Inc, Beijing, China) for 30 min on ice, and then centrifuged (12,000  g) for 15 min at 4 C. Protein concentration in the lysate was determined using a BCA protein assay kit (Vazyme Biotech, Nanjing, China). Exactly 20 mg of protein was separated on a 12% SDS–PAGE gel and then transferred onto a polyvinylidene fluoride membrane (Merck Millipore). The membrane was blocked using 5% nonfat dried milk in Trisbuffered saline containing 0.1% Tween 20 (pH 7.6) for 1 h at room temperature and then incubated overnight at 4 C with primary antibodies, namely, rabbit anti-DCN polyclonal antibody (1:300, Bioss, bs-1695R, Beijing, China), rabbit anti-Bcl-2 polyclonal antibody (1:1000, Beyotime Biotechnology, AB112, Jiangsu, China), mouse anti-Bax monoclonal antibody (1:1000, Beyotime Biotechnology, AA026, Jiangsu, China), mouse anti-p21 monoclonal antibody (1:300, Beyotime Biotechnology, AP021, Jiangsu, China), or mouse anti–actin monoclonal antibody (1:1000, Beyotime Biotechnology, AA128, Jiangsu, China). The membrane was incubated with an HRP-conjugated secondary antibody for 1 h at room temperature. The HRP was subsequently detected by an enhanced chemiluminescence detection system (BeyoECL Star, Beyotime Biotechnology, P0018 A, Jiangsu, China). 2.9. Statistical analysis

2.7. Flow cytometric analysis of apoptosis and cell cycle Apoptosis was detected using an Annexin V-FITC/PI Apoptosis Detection Kit (MultiSciences Biotech, Hangzhou, China) according to manufacturer’s protocol. Briefly, freshly

All data were presented as means  SE. The results were analyzed by using 1- or 2-way ANOVA followed by an LSD post hoc test or by using Student’s t test to compare the 2 groups. A P value of <0.05 was considered statistically

J.Y. Peng et al. / Domestic Animal Endocrinology 57 (2016) 108–116

significant. All statistical analyses were performed using SPSS 17.0. 3. Results 3.1. Cloning of DCN cDNA A 1,522 bp full-length goat DCN cDNA was obtained from a goat ovary through RT-PCR. The cDNA of the goat DCN (HQ326237) contains an ORF of 1,083 bp encoding a 360residue protein, along with a 16-residue putative signal peptide and a 344-residue putative mature peptide (Fig. 1). A potential glycosaminoglycan (GAG) attachment site at

111

Ser34-Gly35 was followed by a cysteine-rich region containing 4 cysteine residues. The DCN protein was predicted to contain 5 tandem LRRs and 4 N-glycosylation sites (Asn212, Asn247, Asn263, and Asn304; Fig. 2A). Moreover, the amino acid sequences of the goat DCN protein was aligned with the known amino acid sequences of their counterparts in other species by using ClustalW. Homology analyses indicated that the DCN protein of goat shares 75%, 79%, 89%, 94%, 95%, 99%, and 99% sequence similarity with the DCN of Rattus norvegicus, Mus musculus, Homo sapiens, Equus caballus, Sus scrofa, Bos taurus, and Ovis aries, respectively (Table 1). The sequence similarity results were further reflected in a phylogenetic tree constructed using the neighbor-joining method (Fig. 2B). The

Fig. 1. Nucleotide and deduced amino acid sequences of goat DCN. The putative signal peptide was shaded in gray. Nucleotides were numbered at the right end of the lines. The break-underlined Ser–Gly dipeptide represented potential GAG attachment sites. Cysteine residues were indicated by circles. The leucine-rich repeats (LRR, xL-x-x-L-x-L/I-x-x-N-x-L/I) were underlined. The translation start codon (ATG) and stop codon (TGA) were bold typed. A, adenine; DCN, decorin; G, guanine; T, thymine.

112

J.Y. Peng et al. / Domestic Animal Endocrinology 57 (2016) 108–116

Fig. 2. Multiple sequence alignment and phylogenic tree of DCN. (A) Multiple sequence alignment of DCN amino acids of cattle, sheep, goat, swine, horse, human, mouse, and rat. The putative signal peptide was in solid frame. Filled diamonds indicate the putative N-linked oligosaccharide attachment sites, while arrows indicate the cysteine residues of the mature protein. The amino acid sequences were obtained from the NCBI GenBank database (accession numbers: Bos taurus, NP_776331; Ovis aries, NP_001009218; Sus scrofa, NP_999085; Equus caballus, NP_001075394; Homo sapiens, NP_001911; Mus musculus, NP_001177380; Rattus norvegicus, NP_077043). (B) The phylogenic tree was constructed using MEGA 5.0 program with neighbor-joining method and bootstrap resampling (1,000 replications). The numbers in this tree indicated the bootstrap value (%). DCN, decorin.

phylogenetic analysis showed that DCN is clearly divided into 2 clades: rodents and other mammals. Moreover, the goat DCN is closely related to sheep and cattle DCN. 3.2. Analysis of DCN gene expression in various goat tissues The relative expression of DCN mRNA in various goat tissues was studied by RT PCR. Our analysis showed that all

the 11 tissues expressed DCN mRNA. The highest DCN expression levels were detected in the uterus and consecutively followed by the mammary gland, oviduct, lung, fat, and ovary (Fig. 3A). To further establish the expression pattern of DCN in goat ovary, we performed immunohistochemistry analysis. The results demonstrated that DCN protein was present in the GCs and oocytes from all types of follicles and TCs of

J.Y. Peng et al. / Domestic Animal Endocrinology 57 (2016) 108–116 Table 1 Percentage of sequence identity at nucleotide and amino acid sequence of goat DCN. Species

Accession no.

Nucleotide identity (%)

Amino acid identity (%)

Ovis aries

NM_001009218/ NP_001009218 NM_173906/NP_776331 NM_213920/NP_999085 NM_001081925/ NP_001075394 NM_001920/NP_001911 NM_001190450/ NP_001177380 NM_024129/NP_077043

99

99

98 93 92

99 95 94

89 78

89 79

77

75

Bos taurus Sus scrofa Equus caballus Homo sapiens Mus musculus Rattus norvegicus

antral follicles (Fig. 3B-F). Apart from follicles, strong immunoreaction to DCN was also observed in corpus luteum (Fig. 3G). For all antibodies tested, control

113

reactions (Fig. 3G) confirmed the absence of nonspecific staining.

3.3. Intracellular signaling mechanism of DCN mRNA induction in vitro Our previous study demonstrated that hCG induces DCN mRNA expression in primary cultures of goat granulosa cells. Thus, we further investigated the regulation mechanism of DCN in response to hCG stimulation. protein kinase A (PKA) and protein kinase C (PKC) signaling pathways are activated by hCG in preovulatory granulosa cells [20]. In this study, the cells were treated with FSK (an activator of adenylate cyclase) or PMA (an activator of protein kinase C) to mimic the activation of PKA and PKC signaling pathways. After 4 h, the treated cells were collected for total RNA isolation. Figure 4A shows that treatments with FSK or PMA increased DCN mRNA expression in cultured granulosa cells of goat.

Fig. 3. Expression of DCN in goat various tissues and ovary. (A) The expression of DCN mRNA was measured in various tissues by real-time PCR. Levels of mRNA for DCN were normalized to the b-actin in each sample (mean  SEM; n ¼ 4 biological replicates). (B–G) Immunohistochemistry for DCN protein in the goat ovary illustrates: (B) primordial follicle, (C) primary follicle, (D) secondary follicle, (E) small antral follicle, (F) granulosa and theca cells from a large antral follicle, (G) corpus luteum and negative control. Scale bars represent 50 mm. DCN, decorin.

114

J.Y. Peng et al. / Domestic Animal Endocrinology 57 (2016) 108–116

Fig. 4. Regulation of DCN mRNA expression by agonists or inhibitors of various intracellular signaling modulators in granulosa cells in vitro. (A) Real-time PCR analysis showed the expression of DCN in granulosa cells from goat ovaries cultured in medium alone (DMSO) or with hCG (1 IU/mL), forskolin (FSK, 10 mM), PMA (100 nM) for 4 h. Relative levels of DCN mRNA were normalized to the b-actin mRNA in each sample (mean  SEM; n ¼ 3 independent culture experiments). (B) Granulosa cells were cultured with medium alone (DMSO), inhibitors of various signaling molecules (an inhibitor of PKA [H89, 10 mM], p38 kinase [SB2035850 {SB}, 20 mM], PI3 kinase [LY294002 {LY}, 25 mM]) hCG, or hCG þ inhibitors of various signaling molecules for 4 h (B). Levels of DCN mRNA were measured by realtime PCR. Relative levels of DCN mRNA were normalized to the b-actin mRNA in each sample (mean  SEM; n ¼ 3 independent culture experiments). Bars with no common letters were significantly different (P < 0.05). DCN, decorin; DMSO, dimethylsulfoxide; PMA, phorbol 12-myristate 13-acetate.

To further determine the signaling mediators involved in hCG-induced DCN expression, we cultured the granulosa cells with or without hCG (1 IU/mL) under 1 h pretreatment with 0.1% DMSO, PKA inhibitor (H89, 10 mM), PKC inhibitor (GF109203X [GF], 1 mM), PI3K inhibitor (LY294002 [LY], 25 mM), or p38 MAPK inhibitor (SB2035850 [SB], 20 mM) for 4 h. The stimulatory effect of hCG on DCN mRNA was reduced by treatment with specific inhibitors of PKA, MAPK kinase, PI3K, and p38 kinase (Fig. 4B).

3.4. Effects of DCN on granulosa cell apoptosis and cell cycle To determine whether overexpression of DCN affects granulosa cell apoptosis in vitro, we transfected the goat granulosa cells with pcDNA3.1 empty vector or pcDNA3.1DCN for 24 h. The cells were then harvested and detected by flow cytometry using an Annexin V-FITC/PI apoptosis kit. Figure 5A–C shows that pcDNA3.1-DCN effectively increased the stimulated DCN expression both at mRNA and protein levels in granulosa cells compared with those in pcDNA3.1 empty vector-treated cells. Moreover, the percentage of apoptotic cells was significantly increased in the pcDNA3.1-DCN-treated cells (2.71%), as compared with the group treated with the negative control pcDNA3.1 empty vector (1.56%; Fig. 5D). To examine whether DCN was also associated with cell cycle of granulosa cells, we analyzed the cell cycle distribution of the granulosa cells transfected with pcDNA3.1 empty vector or with pcDNA3.1-DCN for 24 h. As shown in Figure 5E, the ratios of cells in the G0/G1 phases significantly increased in the pcDNA3.1-DCN group compared with those in the empty vector group. However, the ratios

of cells in the S phases significantly decreased in the pcDNA3.1-DCN group compared with those in the empty vector group. No significant difference in G2 phases was observed between the pcDNA3.1-DCN and empty vector groups. 3.5. Effects of DCN on expression of Bax, Bcl-2, and p21 protein To gain some insight into the molecular mechanism of the cell-inhibiting effect of DCN, we further investigated the protein expression levels of Bcl-2, Bax, and p21 in vitro. As shown in Figure 5B and C, pcDNA3.1-DCN treatment significantly enhanced the expression of p21, whereas no significant change was observed in Bax and Bcl-2 protein expression levels. No significant difference in the Bax/Bcl-2 ratio was also observed (data not shown). 4. Discussion The present study reports on the cloning and characterization of DCN cDNA obtained from goat and reveal the first complete sequence of DCN of this important species. The result of multiple sequence alignment showed that the amino acid sequences of DCN are highly conserved in various mammals. The deduced amino acid sequence showed 99%, 99%, 95%, 94%, 89%, 79%, and 75% similar homologs with sheep, cattle, pig, horse, human, mouse, and rat, respectively. Sequence analysis revealed that the protein contains a signal peptide with 16 amino acid residues that direct the nascent protein to the rough endoplasmic reticulum; a propeptide of 14 amino acids with unknown

J.Y. Peng et al. / Domestic Animal Endocrinology 57 (2016) 108–116

115

Fig. 5. Overexpression of DCN increased apoptosis and reduced viability of granulosa cells. (A) The effect of pcDNA-DCN on the levels of DCN mRNA and protein as revealed by real-time PCR. Relative level of DCN mRNA was normalized to the b-actin in each sample (mean  SEM; n ¼ 3 independent culture experiments). *P < 0.05. (B, C) Western blot analysis showed the expression of DCN, Bax, Bcl-2, and p21 in granulosa cells from goat ovaries cultured with control pcDNA3.1 empty vector or pcDNA3.1-DCN for 24 h. Relative level of DCN protein was normalized to the b-actin in each sample (mean  SEM; n ¼ 3 independent culture experiments). (D) Apoptosis and (E) cell cycle distribution was measured using the flow cytometric analysis in granulosa cells cultured with control pcDNA3.1 empty vector or pcDNA3.1-DCN for 24 h (mean  SEM; n ¼ 3 independent culture experiments). DCN, decorin.

function; the glycosaminoglycan-attachment region; 4 highly conserved cysteine residues (CX3CXCX6C) that are characteristic of the class I SLRPs; and a protein core, including five LRRs. Earlier studies have demonstrated that DCN is widely expressed in many mammalian tissues, including the uterus [10], mammary gland [21], kidney [22], and ovary [16]. In both mouse and sheep, high levels of DCN were detected in the uterus, and treatment with E2 and P4 upregulated DCN mRNA and protein levels [10,11]. In the mammary gland of pregnant women, DCN expression is significantly lower in the luteal phase than in the follicular phase [21]. Consistent with these observations, our results showed high DCN mRNA levels in the uterus and mammary gland suggesting a potential role that DCN plays in these tissues. Moreover, a recent study demonstrated that, in the human and monkey ovary, DCN is produced from different cells including stromal cell, follicular theca cells, and luteinized granulosa cells [16]. This observation is also consistent with our findings wherein high DCN levels were detected in goat ovary. LH surge is responsible for the final stage of oocyte maturation and induces expression of ovulation-related genes by activating a number of kinases, including PKA, PI3K, and RTKs [20]. Our previous study demonstrated that

hCG induced a rapid and transient expression of DCN mRNA in cultured granulosa cells of goat [17]. The present study further investigated the LH-induced signaling pathway involved in DCN expression in granulosa cells. We found that DCN expression in granulosa cells was downregulated by hCG-induced activation of PKA, PKC, PI3K, and p38 kinase. These results suggested that DCN regulation in granulosa cells is mediated by multiple signaling pathways, including the PKA-dependent pathway (cAMP/PKA/MEK) and the PKA- and PKC-independent pathways (p38 kinase and PI3K). DCN is a multifunctional protein that stimulates cell cycle, proliferation, and apoptosis of various cell types [3,23–25]. Yamaguchi and Ruoslahti [15] reported that overexpression of DCN in the ovary cells of Chinese hamster suppresses cell proliferation. Transfection of human DCN into liver HepG2 carcinoma cells causes apoptosis and arrests cell growth [25]. In addition, overexpression of DCN induces apoptosis and cell growth arrest in rat mesangial cells [23]. Consistent with these observations, our results showed that overexpression of DCN resulted in apoptosis and arrested cell growth in the G0/G1 phase of the cultured granulosa cells in vitro. Two major apoptosis pathways have been defined: the death receptor

116

J.Y. Peng et al. / Domestic Animal Endocrinology 57 (2016) 108–116

pathway and the mitochondria pathway [26]. Bcl-2 and Bax genes control the mitochondrial apoptosis pathway. Bcl-2 is an antiapoptotic protein, whereas Bax exerts proapoptosis effect by antagonizing Bcl-2 [27,28]. Therefore, the ratio of Bcl-2/Bax expression is an indication of apoptosis. This study also investigated the effect of DCN on these apoptosis-related proteins. We found that in granulosa cells, DCN treatment exerted no effects on Bax and Bcl-2 expression and on Bcl-2/Bax ratio. These findings suggested that the inhibitory effect of DCN on goat granulosa cells is possibly not caused by activation of the mitochondrial apoptosis pathways. p21, also known as cyclin-dependent kinase inhibitor 1A, is an important cyclin-dependent kinase inhibitor that induces cell cycle arrest and inhibits cell proliferation [29,30]. Many studies have shown that DCN upregulates p21 expression and subsequently arrests cells in the G1 stage of the cell cycle [23,25,31]. Our results suggested that overexpression of DCN inhibited granulosa cell growth by promoting p21 expression. This result indicates that the mechanism of DCN in suppressing granulosa cell proliferation is similar to that in other cells. In summary, full-length cDNA of goat DCN was cloned. DCN is widely expressed in adult goat tissues and is highly expressed in the uterus. In addition, our results demonstrated that the hCG-dependent induction of DCN expression was mediated through multiple signaling pathways, including the PKA, p38 kinase, and PI3K pathways. The present study also confirmed the effect of DCN on granulosa cells, and our results suggested that DCN promotes cell apoptosis via a nonmitochondrial apoptosis pathway and induces cell growth arrest by upregulating expression of p21. Further studies are needed to determine the precise molecular mechanism of the gonadotropin-dependent regulation of DCN and its functional roles in follicular development in goats. Acknowledgments This work was supported by the National Support Program of China (2011BAD28B05-3), the Science and Technology Innovation Project of Shaanxi Province (2011KTCL02-09), and the National Spark Plan (2013GA850003). The authors have nothing to disclose. References [1] Lu P, Takai K, Weaver VM, Werb Z. Extracellular matrix degradation and remodeling in development and disease. Cold Spring Harb Perspect Biol 2011;3. [2] Schaefer L, Iozzo RV. Biological functions of the small leucine-rich proteoglycans: from genetics to signal transduction. J Biol Chem 2008;283:21305–9. [3] Shi X, Liang W, Yang W, Xia R, Song Y. Decorin is responsible for progression of non-small-cell lung cancer by promoting cell proliferation and metastasis. Tumour Biol 2015;36:3345–54. [4] Chen S, Birk DE. Focus on molecules: decorin. Exp Eye Res 2011;92: 444–5. [5] Hausser H, Groning A, Hasilik A, Schonherr E, Kresse H. Selective inactivity of TGF-beta/decorin complexes. FEBS Lett 1994;353:243–5. [6] Takeuchi Y, Kodama Y, Matsumoto T. Bone matrix decorin binds transforming growth factor-beta and enhances its bioactivity. J Biol Chem 1994;269:32634–8.

[7] Iozzo RV, Moscatello DK, McQuillan DJ, Eichstetter I. Decorin is a biological ligand for the epidermal growth factor receptor. J Biol Chem 1999;274:4489–92. [8] Iozzo RV, Buraschi S, Genua M, Xu SQ, Solomides CC, Peiper SC, Gomella LG, Owens RC, Morrione A. Decorin antagonizes IGF receptor I (IGF-IR) function by interfering with IGF-IR activity and attenuating downstream signaling. J Biol Chem 2011;286:34712–21. [9] Goldoni S, Humphries A, Nystrom A, Sattar S, Owens RT, McQuillan DJ, Ireton K, Iozzo RV. Decorin is a novel antagonistic ligand of the met receptor. J Cell Biol 2009;185:743–54. [10] Salgado RM, Favaro RR, Zorn TM. Modulation of small leucine-rich proteoglycans (SLRPs) expression in the mouse uterus by estradiol and progesterone. Reprod Biol Endocrinol 2011;9:22. [11] Wu WX, Zhang Q, Unno N, Derks JB, Nathanielsz PW. Characterization of decorin mRNA in pregnant intrauterine tissues of the ewe and regulation by steroids. Am J Physiol Cell Physiol 2000;278: C199–206. [12] Ricciardelli C, Rodgers RJ. Extracellular matrix of ovarian tumors. Semin Reprod Med 2006;24:270–82. [13] Grisaru D, Hauspy J, Prasad M, Albert M, Murphy KJ, Covens A, Macgregor PF, Rosen B. Microarray expression identification of differentially expressed genes in serous epithelial ovarian cancer compared with bulk normal ovarian tissue and ovarian surface scrapings. Oncol Rep 2007;18:1347–56. [14] Nivet AL, Vigneault C, Blondin P, Sirard MA. Changes in granulosa cells’ gene expression associated with increased oocyte competence in bovine. Reproduction 2013;145:555–65. [15] Yamaguchi Y, Ruoslahti E. Expression of human proteoglycan in Chinese hamster ovary cells inhibits cell proliferation. Nature 1988; 336:244–6. [16] Adam M, Saller S, Strobl S, Hennebold JD, Dissen GA, Ojeda SR, Stouffer RL, Berg D, Berg U, Mayerhofer A. Decorin is a part of the ovarian extracellular matrix in primates and may act as a signaling molecule. Hum Reprod 2012;27:3249–58. [17] Peng J, Xin H, Han P, Gao K, Gao T, Lei Y, Ji S, An X, Cao B. Expression and regulative function of tissue inhibitor of metalloproteinase 3 in the goat ovary and its role in cultured granulosa cells. Mol Cell Endocrinol 2015;412:104–15. [18] Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. Mega5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 2011;28:2731–9. [19] Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 2001;25:402–8. [20] Jo M, Curry Jr TE. Luteinizing hormone-induced RUNX1 regulates the expression of genes in granulosa cells of rat periovulatory follicles. Mol Endocrinol 2006;20:2156–72. [21] Hallberg G, Andersson E, Naessen T, Ordeberg GE. The expression of syndecan-1, syndecan-4 and decorin in healthy human breast tissue during the menstrual cycle. Reprod Biol Endocrinol 2010;8:35. [22] Schaefer L. Small leucine-rich proteoglycans in kidney disease. J Am Soc Nephrol 2011;22:1200–7. [23] Wu H, Wang S, Xue A, Liu Y, Liu Y, Wang H, Chen Q, Guo M, Zhang Z. Overexpression of decorin induces apoptosis and cell growth arrest in cultured rat mesangial cells in vitro. Nephrology (Carlton) 2008; 13:607–15. [24] Ferdous Z, Peterson SB, Tseng H, Anderson DK, Iozzo RV, GrandeAllen KJ. A role for decorin in controlling proliferation, adhesion, and migration of murine embryonic fibroblasts. J Biomed Mater Res A 2010;93:419–28. [25] Zhang Y, Wang Y, Du Z, Wang Q, Wu M, Wang X, Wang L, Cao L, Hamid AS, Zhang G. Recombinant human decorin suppresses liver HepG2 carcinoma cells by p21 upregulation. Onco Targets Ther 2012;5:143–52. [26] Gross A, McDonnell JM, Korsmeyer SJ. BCL-2 family members and the mitochondria in apoptosis. Genes Dev 1999;13:1899–911. [27] Adams JM, Cory S. The Bcl-2 protein family: arbiters of cell survival. Science 1998;281:1322–6. [28] Mackey TJ, Borkowski A, Amin P, Jacobs SC, Kyprianou N. Bcl-2/bax ratio as a predictive marker for therapeutic response to radiotherapy in patients with prostate cancer. Urology 1998;52:1085–90. [29] Xiong Y, Hannon GJ, Zhang H, Casso D, Kobayashi R, Beach D. P21 is a universal inhibitor of cyclin kinases. Nature 1993;366:701–4. [30] Abbas T, Dutta A. P21 in cancer: intricate networks and multiple activities. Nat Rev Cancer 2009;9:400–14. [31] Santra M, Skorski T, Calabretta B, Lattime EC, Iozzo RV. De novo decorin gene expression suppresses the malignant phenotype in human colon cancer cells. Proc Natl Acad Sci U S A 1995;92:7016–20.