STAT3 Inhibits Cell Proliferation at Least in Part Via Directly Negatively Regulating FST Gene Expression


 Background: Follistatin (FST) is a secretory glycoprotein and belongs to the TGF-β superfamily. Previously, we found that two single nucleotide polymorphisms (SNPs) of sheep FST gene were significantly associated with wool quality traits in Chinese Merino sheep (Junken type), indicating that FST is involved in the regulation of hair follicle development and hair trait formation. The transcription regulation of human and mouse FST genes has been widely investigated, and many transcription factors have been identified to regulate FST gene, such as erythroid 2-related factor 2 (Nrf2), Estrogen-related receptor-β (ERRβ), β-catenin/transcription factor 4 (TCF4) and transcription factor Sp1. However, to date, the transcriptional regulation of sheep FST is largely unknown. The objective of this study was to investigate the transcriptional regulation of sheep FST gene in hair follicles. Results: Genome walking analysis revealed that the gap region upstream of sheep genomic FST gene was 775 bp long. Transcription factor binding site analysis showed sheep FST promoter region contained a conserved putative binding site for signal transducer and activator of transcription 3 (STAT3), located at nucleotides -423 to -416 relative to the first nucleotide (A, +1) of the initiation codon (ATG). The dual-luciferase reporter assays showed that STAT3 inhibited the activity of the FST promoter reporter, and the mutation of the putative STAT3 binding site attenuated the inhibitory effect of STAT3 on the FST promoter activity. Furthermore, chromatin immunoprecipitation assay (ChIP) indicated that STAT3 directly binds to the FST promoter. The further functional study displayed that FST and STAT3 played opposite roles in cell proliferation. Overexpression of FST significantly promoted the proliferation of sheep fetal fibroblasts (SFFs) and human keratinocyte (HaCaT) cells, and overexpression of STAT3 significantly inhibited the proliferation of SFFs and HaCaT cells, which was accompanied by a significantly reduced expression of FST gene (P < 0.05). Conclusions: STAT3 directly negatively regulates sheep FST gene and inhibits cell proliferation. The findings will contribute to understanding molecular mechanisms that underlie hair follicle development and wool trait formation.

unknown. The objective of this study was to investigate the transcriptional regulation of sheep 23 FST gene in hair follicles. 24 Results: Genome walking analysis revealed that the gap region upstream of sheep genomic FST 25 gene was 775 bp long. Transcription factor binding site analysis showed sheep FST promoter 26 region contained a conserved putative binding site for signal transducer and activator of 27 transcription 3 (STAT3), located at nucleotides -423 to -416 relative to the first nucleotide (A, +1) 28 of the initiation codon (ATG). The dual-luciferase reporter assays showed that STAT3 inhibited 29 the activity of the FST promoter reporter, and the mutation of the putative STAT3 binding site 30 attenuated the inhibitory effect of STAT3 on the FST promoter activity. Furthermore, chromatin 31 immunoprecipitation assay (ChIP) indicated that STAT3 directly binds to the FST promoter. The 32 further functional study displayed that FST and STAT3 played opposite roles in cell proliferation. 33 Background 42 Wool is the product of hair follicles and wool traits are influenced by hair follicles. The hair 43 follicle is a skin appendage with a complex structure composed of the dermal papilla, hair bulbs, 44 outer root sheaths (ORS), inner root sheaths (IRS), and the hair matrix [1,2]. Hair follicle 45 morphogenesis and development involve proliferation, differentiation and apoptosis of hair 46 follicle stem cells [2,3]. The hair follicle undergoes life-long cyclic transformations exhibiting 47 opposite directions with two pairs of primers FST-P3 and FST-P4 (Table 1) using the sheep  131 genomic DNA as the template. Subsequently, the amplified FST promoter fragments were inserted 132 into the Kpn I and Hind III site of pGL3-basic (Promega, United States) to yield two FST 133 promoter reporters. The reporter with the FST promoter fragment in the right direction was named 134 pFST (-980/-340) and the other one with the FST promoter fragment in opposite direction was 135 named pFST (-340/-980 collected by centrifugation at 10, 000 × g for 5 min at 4℃. The equal amounts of protein from the 154 cell lysates were separated by 12% SDS-polyacrylamide gel electrophoresis and transferred to 155 nitrocellulose membranes (Millipore, United States). The blots were blocked in PBS containing 156 5% (w/v) dry milk and 0.1% Tween 20 for 2 h and then incubated with primary antibody dilution 157 buffer (Beyotime Biotechnology, China) containing Myc-tag mouse monoclonal antibody (Abcam, 158 1:1,000) at room temperature for 2 h. After washing with PBS three times, the blots were 159 incubated with a secondary antibody dilution buffer containing horseradish peroxidase-conjugated 160 anti-mouse secondary antibody (Abcam, 1:5,000) for 1 h, followed by washing three times with 161 PBS. The blots were visualized using an ECL Plus detection kit (Beyotime Biotechnology, China). 162

Statistical analysis 197
Data were expressed as the mean ± SE and analyzed with SAS 9.1.3 (SAS Institute Inc., NC). The 198 student's t-test was used to examine the significance of the difference in gene expression. 199 Statistical significance was indicated by *P < 0.05, **P < 0.01. 200

Results 201
Sheep FST promoter contains a conserved STAT3 binding site 202 There is a genomic gap immediate upstream of sheep FST gene according to the reference genome 203 sheep FST gene, such as homeobox A4 (HOXA4), E2F transcription factor 2 (E2F2), hepatocyte 213 nuclear factor 4 (HNF4), and STAT3. Of these transcription factors, STAT3 interested us. As 214 shown in Figure 1A, the putative STAT3 binding sites were conserved among various species. 215 STAT3 has been well defined as a key transcription factor for cell signal activation and 216 transduction and plays critical roles in various biological activities including cell proliferation, 217 migration, survival and oncogenesis [23,33]. Furthermore, several independent lines of evidence indicated that STAT3 is implicated in hair follicle morphogenesis and development [34,35]. These 219 data led us to speculate that STAT3 may directly regulate FST gene in hair follicle development. 220

STAT3 inhibits the FST promoter activity 221
To test the speculation whether STAT3 directly regulates FST gene expression, firstly, we 222 constructed and verified the STAT3 expression vector, pCMV-Myc-STAT3 by western blotting 223 ( Figure 1B). A highly conserved region (-980/-340) of sheep FST promoter, which contained the 224 putative conserved STAT3 binding site, was amplified and cloned into pGL3-basic vector in 225 opposite direction, respectively, yielding the FST promoter reporters: pFST(980/-340) and 226 pFST(-340/-980) as a negative control. In addition, the FST promoter reporter with mutation of the 227 STAT3 binding site, named pFST(-980/-340)-mutSTAT3, was constructed. The reporter gene 228 assay showed that, as expected, both pGL3-basic vector and pFST(-340/-980) as a negative 229 control had very lower luciferase activity, and no difference in luciferase activity was observed 230 between them (P > 0.05, Figure 1C). The luciferase activity of pFST(-980/-340) and 231 pFST(-980/-340)-mutSTAT3 plasmids were 3.39-and 6.23-fold higher than that of the pGL3-basic 232 vector (P < 0.05, Figure 1C), suggesting that the -980/-340 FST promoter region has promoter 233 activity and that mutation of STAT3 binding site increases the promoter activity of FST gene. 234 Co-transfection analysis showed that the luciferase activity of pFST(-980/-340) was significantly 235 reduced by 22.83% in the cells co-transfected with pCMV-Myc-STAT3, as compared with the 236 cells co-transfected with pCMV-Myc (P < 0.05, Figure 1D). Intriguingly, the luciferase activity of 237 pFST(-980/-340)-mutSTAT3 was also significantly reduced in the cell co-transfected with 238 pCMV-Myc-STAT3, as compared with the cells co-transfected with pCMV-Myc (P < 0.05, Figure  239 1D). Taken together, these data suggested that STAT3 inhibits sheep FST promoter activity. 8.89-fold, respectively) in the DNA immunoprecipitated by the Myc-specific antibody compared with the negative controls (mouse IgG, A and B) (P < 0.05, Figure 1E). Consistent with the 249 ChIP-qPCR results, agarose gel electrophoresis showed that, compared with the negative control 250 (mouse IgG, A, and B), more PCR products (-547/-356 region of FST promoter) were obtained 251 from the DNA fragments immunoprecipitated by the Myc-specific antibody ( Figure 1F). In 252 summary, these data indicated that STAT3 directly binds to and regulates the FST promoter.   Figure 3E and F).
Taken together, these data not only support that STAT3 negatively regulates FST gene expression, 277 but also indicate that STAT3 inhibits cell proliferation at least in part via directly downregulating 278

FST gene expression. 279
Discussion 280 In the present study, we demonstrated that STAT3 directly negatively regulates sheep FST gene. 281 Our evidences are as follows: (1) Bioinformatics analysis showed that FST promoter harbored a 282 conserved putative STAT3 binding site ( Figure 1A ). (2) The luciferase reporter assay showed that 283 mutation of STAT3 binding site led to an increase in the FST promoter activity and that STAT3 284 inhibited the FST promoter activity ( Figure 1C  with pCMV-Myc (P < 0.05, Figure 1D). This may be dual to several reasons. Firstly, STAT3 may 301 bind to its noncanonical binding sites in this FST promoter and inhibit FST promoter activity. 302 Secondly, STAT3 may indirectly regulate FST promoter activity through regulation of the 303 expression of the transcription factors which have binding sites in FST promoter. Lastly, STAT3 304 may indirectly regulate FST promoter activity by protein interaction with some transcription 305 factors, which have binding sites in FST promoter. Further study is required to determine the 306 precise mechanism underlying the partial inhibitory effect of STAT3 on the reporter 307 pFST(-980/-340)-mutSTAT3 in the future. 308 In the present study, we demonstrated that sheep FST overexpression promoted SFFs and HaCaT 309 cell proliferation ( Figure 2B-E Immunoprecipitated DNA was quantified by qRT-PCR using the specific pair of primers (Table 1), 362 which was designed to amplify the -547/-356 region of the FST promoter. The SFFs and HaCaT cells were seeded in 96-well plates (5 × 10 4 cells / well) and transfected