Expression of Porcine SFRP4 and Its Effect on Differentiation of Preadipocytes via Inhibitor SP600125

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1 ISSN CN / Q http / / cjbmb bjmu edu cn Chinese Journal of Biochemistry and Molecular Biology ~ 463 SFRP4 SP * secreted frizzled-related protein 4 SFRP4 Wnt Solexa PCR RT-qPCR SFRP4 JNK SP JNK SFRP4 mrna SFRP4 P < 0 01 SFRP4 SFRP4 SP PPARγ FABP4 ATGL Perilipin P < 0 01 SFRP4 4 SP Q78 Q246 Expression of Porcine SFRP4 and Its Effect on Differentiation of Preadipocytes via Inhibitor SP GUAN Hua ZHENG Jia-Meng CHEN Zong-Zheng SONG Zi-Yi YANG Hao YANG Gong-She * Laboratory of Animal Fat Deposition and Muscle Development College of Animal Science and Technology Northwest A&F University Yangling Shaanxi China Abstract Secreted frizzled-related protein 4 SFRP4 was the regulator of Wnt signaling pathway The expression of SFRP4 in fat tissues of different genotype pigs and in different growth periods were explored by high-throughout sequencing Solexa and real-time quantity PCR RT-qPCR The effect on differentiation of preadipocyte and the expression level of SFRP4 were explored by inhibitor SP in JNK signaling pathway The results showed that expression level of SFRP4 was higher in fat tissues of obese-type than that in the lean-type Expression level of SFRP4 in the porcine fat tissue and adipocyte was influenced by ages and was higher in fat but lower in other tissues SP promoted the adiocytes to differentiate and significantly increased expression levels of PPARγ FABP4 ATGL Perilipin but the SFRP4 expression level was inhibited significantly This research provides a new reference for screen the key genes in adipocyte differentiation Key words secreted frizzled-related protein 4 high-throughput sequencing preadipocyte inhibitor SP differentiation No 2009ZX B * Tel E- mail gsyang999@ hotmail com Received January Accepted March Supported by Genetically Modified Organisms Breeding Major Projects No 2009ZX B * Corresponding author Tel E- mail gsyang999@ hotmail com

2 secreted frizzled- related protein 4 SFRP Rattner SFRP4 1 Wnt 15 Wnt / cm 2 SFRP4 3 4 ~ 9 60 mm 10% 10 DMEM / F % CO 2 1 d 2 d 1 SFRP4 Fzd5 Wnt7a 100% 15 μmol / L Wnt7a JNK SP h 48 h 72 h 11 3 DMSO 3 JNK RNA 12 ~ 13 JNK SFRP Ouchi 16 PBS I SFRP min r / min 5 min SFRP4 adipocyte AD PCR AD SVF RNA RT-qPCR SFRP4 SFRP4 mrna mrna 1 4 Solexa 17 JNK RNA 1 ~ 2 g Oligo dt beads SP JNK RNA mrna cdna 4 JNK SFRP4 Nla III cdna Illumina ~ adapter 2 5 kg 0 5% 0 5 hn 1 Clean Tag Clean tag Clean tag Clean tag Trizol RT TaKaRa RTqPCR DMEM / 1 5 PCR F12 I Gibco DMEM / F12 Hyclone PVDF ECUL Invtrogen SFRP4 anti-goat β-actin p-jnk anti-mouse Santa Cruz JNK SP Sigma stromal vascular fractions SVF adapter1 Mme I 3' 20 3 ' Illumina adapter2 Primer GX1 Primer GX2 PCR 6% TBE PAGE 85 Illumina Clean tag β-actin SFRP4 PPARγ FABP4 ATGL Perilipin RT-qPCR NCBI Table 1

3 5 SFRP4 SP Table 1 Primer sequences and parameters for RT- qpcr amplification of the related genes Gene GenBank accession number Primer sequence 5'-3' Length / bp β-actin AF FP GGACTTCGAGCAGGAGATGG 138 RP AGGAAGGAGGGCTGGAAGAG SFRP4 NM_ FP TGCCAGTGTCCTCACATCCTAC 168 RP GACTGTCCTCTGCTGCTCCTG PPARγ NM_ FP AGGACTACCAAAGTGCCATCAA 142 RP GAGGCTTTATCCCCACAGACAC Perilipin NM_ RP CAGCCAAGGAAGAGTCAGC 107 FR CCTGGAAGGTGTGTTGAGAG FABP4 NM_ RP GAGCACCATAACCTTAGATGG 121 FP AAATTCTGGTAGCCGTGACA ATGL NM_ RP CCTCATTCCACCTGCTCTCC 90 FP GTGATGGTGCTCTTGAGTTCGT - 70 SP DMSO RNA 1 8 cdna SPSS18 0 One-way 1 RT cdna ANOVA SYBR Green I ± Mean ± SE PCR 2 20 μl cdna 1 μl FP RP 1 μl ReaulMasterMix / SYBR solution 9 μl ddh 2 O 20 μl PCR 95 3 min s s s Solexa SFRP4 mrna C t β-actin SFRP4 PPARγ FABP4 Perilipin ATGL PCR Fig 1A CT PCR β-actin 1 6 O PBS 3 4% O min SFRP4 1 7 Western mrna SFRP4 Fig 2A PCR 100 mg 1 ml ml 2 1 SFRP4 SFRP4 SFRP4 mrna Fig 1B min PBS 10 min SFRP4 Solexa 180 SFRP4 mrna SFRP4 PBS μl Fig 2B 2 3 SFRP4 4 PCR r / min 10 min SFRP4 mrna min SFRP4

4 Fig 1 Variation of abundance for SFRP4 in pig fat tissues of different growing phases Two RNA pools of subcutaneous adipose tissue one from 3 days old pigs one from 240 days pigs were used in the expression analysis of SFRP4 All experiments repeated three times Both Solexa sequencing and RT-qPCR analysis revealed that SFRP4 expression level was increased as the age increasing P < 0 01 n = 3 A Expression level of SFRP4 in 3 days old and 240 days old pig fat tissues by Solexa sequencing analysis P < 0 01 n = 3 B Relative expression level of SFRP4 in 3 days old 240 days old pig fat tissues by RT-qPCR P < 0 01 n = 3 Fig 2 Variation of abundance for SFRP4 in different breed pig fat tissues Two RNA pools of subcutaneous adipose tissues one from four 180 days old pigs lean type and one from four 240 days old pigs obese type were used in the expression analysis of SFRP4 All experiments repeated three times Both Solexa sequencing and RT-qPCR analysis revealed that SFRP4 was much higher expressed in obese type porcine fat tissue than in lean type porcine fat tissue P < 0 01 n = 3 A Expression levels of SFRP4 in lean type and obese type porcine fat tissues by Solexa sequencing analysis P < 0 01 n = 3 B Relative expression levels of SFRP4 in lean type and obese type pig fat tissues by RT-qPCR P < 0 01 n = 3 Fig 3A mrna Fig 4C Fig 3B 2 4 SFRP Fig 4D 24 h SFRP4 SFRP4 Fig 4A RT-qPCR Western 80% 0 d 2 d 3 2 d 4 d 6 d 8 d 2 d 0 d 2 d 4 d 6 d 8 d AD Fig 4B 8 d O SVF RT-qPCR SFRP4 mrna

5 5 SFRP4 SP Fig 3 Relative expression levels of SFRP4 in normal pig tissues lean type Collected 180 days various white pig tissues WAT white adipocyte tissue Heart Liver Spleen Lung Kidney S muscle Skeletal muscle and Brain and extracted total RNA and total protein SFRP4 expression levels were determined in different tissues by RT-qPCR and Western blotting and calculated with the 2 - CT method as normalization with β-actin All experiments repeated three times The expression level of SFRP4 is highest in WAT A The mrna expression level of SFRP4 qualified by RT-qPCR n = 3 B Western blot analysized the SFRP4 protein level of various tissues n = 3 Fig 5 Expression of SFRP4 during the pig adipocyte differentiation in vitro Preadipocytes separated from adipose of piglets were directly plated in DMEM / F12 medium differentiated induced and harvested at various time points as indicated extracted total RNA and total protein SFRP4 expression levels were determined by RT-qPCR and - Western blotting and calculated with the 2 CT method as normalization with β-actin All experiments were repeated three times A C RT-qPCR and Western blotting qualified the time course mrna and protein expression levels of SFRP4 Whole cells were harvested at days after inducing differentiation The results indicated that the expression levels of SFRP4 increased gradually reached peak at the 8 days P < 0 05 n = 3 B SFRP4 transcript levels in adipocyte AD and stromal vascular fractions SVF isolated from white adipose tissues of 3 days pig as measured by RT-qPCR analysis and expressed relative to β-actin The SVF mrna expression level of SFRP4 were higher than AD P < 0 05 n = 3 SFRP4 0 d 2 5 JNK Fig 5 A Fig 5C SP JNK SFRP4 mrna SP JNK Fig 5B JNK SFRP4 DMEM / F12 15μmol / L SP600125

6 Fig 4 Morphology observation of porcine primary adipocytes A Adipocytes were cultured in vitro for 24 hours showing stretched fibroblast-like or triangle cell phenotypes B Adipocytes were differentiated in the early stage for 2 days showing small lipid droplet in cytoplasm C Adipocytes were differentiated into mature stage for 8 days Some small lipid droplets fusing into big lipid droplets D Differentiated adipocytes stained by oil red O Lipid droplets were stained red by oil red O 24 h 48 h 72 h 24 h Fig 6A 6D 48 h Fig 6E Fig 6B 72 h Fig 6F Fig 6C SP SFRP4 SP h JNK Fig 6B SFRP4 mrna Fig 7A 7B PPARγ FABP4 Perilipin ATGL mrna 48 h P < 0 01 Fig 7C Fig 6 Morphology observation of porcine primary adipocytes treated with SP Primary cultured porcine preadipocytes were treated with 15 μmol / L SP or DMSO for 72 hours and observed the lipid accumulation by treated with SP or DMSO All experiments were repeated three times The results indicated that SP promote preadipocyte adipogenesis A Treated with DMSO for 24 hours B Treated with DMSO for 48 hours C Treated with DMSO for 72 hours D Treated with SP for 24 hours E Treated with SP for 48 hours F Treated with SP600125for 72 hours JNK mrna SFRP4 SFRP4 3 SFRP4 SFRP4

7 5 SFRP4 SP Fig 7 SP down-regulate SFRP4 mrna levels and promote the porcine preadipocyte differentiation spontaneously Isolated preadipocytes from adipose tissues were directly plated in DMEM / F12 medium when confluenced 100% added 15 μmol / L SP or DMSO control into DMEM / F12 medium and cultured for and 72 hours A Gene expression of SFRP4 was quantified by RT-qPCR and expressed relative to β-actin levels The SFRP4 expression level was decreased significantly as relative to control n = 3 * P < 0 05 * * P < 0 01 B Whole cell extracts were analysized by Western blotting The changes of SFRP4 p-jnk expression were showed in control and SP treated group the SFRP4 and p- JNK protein level were decreased significantly as relative to control C Gene expression of PPARγ FABP4 Perilipin ATGL were quantified by RT-qPCR and expressed relative to β-actin levels The gene expression levels were all increased as relative to control n = 3 * P < 0 05 * * P < 0 01 PCR SFRP4 Yam 20 PCR SFRP4 SFRP4 Northern SFRP4 SFRP4

8 SFRP4 SFRP4 SFRP5 Ouchi 16 II SFRP4 PCR PCR SFRP5 JNK SP Western SFRP4 mrna SFRP5 SFRP4 PPARγ FABP4 SFRP5 ATGL Perilipin SFRP4 JNK SFRP4 SFRP References SFRP4 Wang 30 SFRP4 domain of frizzled receptors J SFRP SFRP4 mrna SFRP4 cancer J Park 31 Suzuki 32 SFRP SFRP4 SFRP2 SFRP4 3T3-L1 SFRP2 33 SFRP SP cancer cells J Cancer Res JNK PCR PPARγ FABP4 ATGL Perilipin mrna JNK PPARγ FABP4 ATGL Perilipin SFRP4 mrna JNK SFRP4 SFRP4 1 Rattner A Hsieh JC Smallwood PM et al A family of secreted proteins contains homology to the cysteine-rich ligand-binding Proc Natl Acad Sci Suzuki H Watkins DN Jair KW et al Epigenetic inactivation of SFRP genes allows constitutive WNT signaling in colorectal Nat Genet Cho HY Choi HJ Sun HJ et al Transgenic mice overexpressing secreted frizzled-related proteins sfrp 4 under the control of serum amyloid P promoter exhibit low bone mass but did not result in disturbed phosphate homeostasis J Bone 4 Stamper B D Park S S Beyer R P et al Differential Expression of Extracellular Matrix-Mediated Pathways in Single- Suture Craniosynostosis J PLoS One e Zhang Y Chen FQ Sun YH et al Effects of DNMT1 silencing on malignant phenotype and methylated gene expression in cervical cancer cells J J Exp Clin Cancer Res Ting AH Suzuki H Cope L et al A requirement for DICER to maintain full promoter CpG island hypermethylation in human 7 Easwaran HP Van Neste L Cope L et al Aberrant silencing of cancer-related genes by CpG hypermethylation occurs independently of their spatial organization in the nucleus J Cancer Res Romaker D Puetz M Teschner S et al Increased expression of secreted frizzled-related protein 4 in polycystic kidneys J J Am Soc Nephrol Jacob F Ukeqjini K Nixdorf S et al Loss of secreted frizzledrelated protein 4 correlates with an aggressive phenotype and

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