Smad4 Feedback Enhances BMPR1B Transcription in Ovine Granulosa Cells

Abstract

BMPR1B is a type 1B receptor of the canonical bone morphogenetic protein (BMP)/Sma- and mad-related protein (Smad) signaling pathway and is well known as the first major gene associated with sheep prolificacy. However, little is known about the transcriptional regulation of the ovine BMPR1B gene. In this study, we identified the ovine BMPR1B gene promoter and demonstrated that its transcription was regulated by Smad4. In sheep ovarian follicles, three transcriptional variants of BMPR1B gene with distinct transcription start sites were identified using 5' RACE assay while variants II and III were more strongly expressed. Luciferase assay showed that the region -405 to -200 nt is the PII promoter region of variant II. Interestingly, two putative Smad4-binding elements (SBEs) were detected in this region. Luciferase and ChIP assay revealed that Smad4 enhances PII promoter activity of the ovine BMPR1B gene by directly interacting with SBE1 motif. Furthermore, in the ovine granulosa cells, Smad4 regulated BMPRIB expression, and BMPRIB-mediated granulosa cells apoptosis. Overall, our findings not only characterized the 5' regulatory region of the ovine BMPR1B gene, but also uncovered a feedback regulatory mechanism of the canonical BMP/Smad signaling pathway and provided an insight into the transcriptional regulation of BMPR1B gene and sheep prolificacy.

Keywords: BMPR1B; Smad4; granulosa cell apoptosis; sheep; transcription factor.

Figure 1

Identification of the transcription start sites of the ovine BMPR1B gene. (A) Schematic diagram showing the different 5′-UTRs of the ovine BMPR1B gene. Exons in the 5′-UTR are shown as long boxes and each transcript is represented by polylines. Exons in the coding region are shown as blue boxes. The translation start site is ATG. P1 is the primer used for 5′-RACE assay. (B) Characteristics of exons on the 5′-UTR of the ovine BMPR1B gene. Nucleotide numbering is relative to +1 at the initiating ATG codon.

Figure 2

Identification of PII core promoter region of the ovine BMPR1B gene. (A) mRNA levels of the transcript variants of the ovine BMPR1B 5′-UTR in follicles of sheep ovary. (B) The schematic diagram of the deletion constructs. The 5′-end of transcript variant II was defined as +1. (C,D) Luciferase assay. The deletion constructs were transfected into KGN cells (C) and HEK293T cells (D), and luciferase activity was measured using a Dual-Luciferase Reporter Assay System. Bars represent the mean ± SEM of at least three repeats. * p < 0.05, ** p < 0.01.

Figure 3

Sequence and putative regulatory elements of the PII core promoter region of the ovine BMPR1B gene. Underline indicates potential binding sites for transcription factors and polyA region. Bold letters represent CG site. Dot represents base deletion.

Figure 4

Smad4 enhances transcription activity of the ovine BMPR1B gene. (A) The SBE motifs were located in the PII promoter of the ovine BMPR1B gene. Two SBE motifs were detected at −299/−296 and −286/−283 nt in the PII core promoter. The 5′-end of transcript variant II was defined as +1. (B) The luciferase reporter constructs of the PII core promoter. PII core promoter region was isolated and subcloned into luciferase reporter vector pGL3-basic. Red letters indicate SBE motif. Green letter indicate mutated SBE motif. (C,D) Smad4 increases PII promoter activity. PII promoter reporter construct was co-transfected with pcDNA3.1-Smad4 into KGN cells (C) and HEK293T cells (D), luciferase activity was detected using a Dual-Luciferase Reporter Assay System. (E,F) Smad4 had no effect on activity of SBEs mutant-type PII promoter in KGN cells (E) and HEK293T cells (F). Bars represent the mean ± SEM of at least three repeats. ** p < 0.01.

Figure 5

Smad4 directly binds to SBE motifs of the ovine BMPR1B promoter region. (A) The luciferase reporter construct of individual SBE motif-mutant type PII promoter. (B,C) Luciferase assay. SBE1 motif-mutant type (B) or SBE2 motif-mutant type (C) PII promoter reporter construct was co-transfected with pcDNA3.1-Smad4 into KGN cells, and luciferase activity was detected using a Dual-Luciferase Reporter Assay System. (D) Ultrasonic time optimization. (E) The location of primers used for ChIP assay. F indicates forward primer. R indicates reverse primer. (F) ChIP assay. Bars represent the mean ± SEM of at least three repeats. ** p < 0.01. ns, no significant.

Figure 6

Smad4 induces endogenous BMPR1B expression in the ovine granulosa cells. Smad4 expression vector pcDNA3.1-Smad4 was transfected into the ovine granulosa cells cultured in vitro, and qRT-PCR and Western blotting was used to quantify mRNA (A) and protein (B) levels, respectively. Bars represent the mean ± SEM of at least three repeats. ** p < 0.01.

Figure 7

Smad4 regulates BMPR1B-mediated cell apoptosis in the ovine granulosa cells. (A) Smad4 suppresses granulosa cell apoptosis. Smad4 expression vector pcDNA3.1-Smad4 was transfected into the ovine granulosa cells cultured in vitro, and fluorescence-activated cell sorting (FACS) was performed to detect cell apoptosis rate. (B) Smad4 inhibits BMPR1B-siRNA-induced granulosa cell apoptosis. pcDNA3.1-Smad4 and BMPR-1B-siRNA were co-transfected into the ovine granulosa cells cultured in vitro, and FACS was performed to detect cell apoptosis rate. Bars represent the mean ± SEM of at least three repeats. ** p < 0.01.