Transforming growth factor-β1 up-regulates connexin43 expression in human granulosa cells

Abstract

Study question: Does transforming growth factor-β1 (TGF-β1) up-regulate connexin43 (Cx43) to promote cell-cell communication in human granulosa cells?

Summary answer: TGF-β1 up-regulates Cx43 and increases gap junction intercellular communication activities (GJIC) in human granulosa cells, and this effect occurs via the activin receptor-like kinase (ALK)5-mediated Sma- and Mad-related protein (SMAD)2/3-SMAD4-dependent pathway.

What is known already: TGF-β1 and its receptors are expressed in human granulosa cells, and follicular fluid contains TGF-β1 protein. In human granulosa cells, Cx43 gap junctions play an important role in the development of follicles and oocytes.

Study design, size, duration: This is an experimental study which was performed over a 1-year period.

Participants/materials, setting, methods: Immortalized human granulosa cells (SVOG cells) and primary human granulosa-lutein cells obtained from women undergoing IVF in an academic research center were used as the study models. Cx43 mRNA and protein expression levels were examined after exposure of SVOG cells to recombinant human TGF-β1. An activin/TGF-β type I receptor inhibitor, SB431542, and small interfering RNAs targeting ALK4, ALK5, SMAD2, SMAD3 and SMAD4 were used to verify the specificity of the effects and to investigate the molecular mechanisms. Real-time-quantitative PCR and western blot analysis were used to detect the specific mRNA and protein levels, respectively. GJIC between SVOG cells were evaluated using a scrape loading and dye transfer assay. Results were analyzed by one-way analysis of variance.

Main results and the role of chance: TGF-β1 treatment increased phosphorylation of SMAD2/3 (P < 0.0001) and up-regulated Cx43 mRNA and protein levels (P < 0.001) in SVOG cells and these stimulatory effects were abolished by the TGF-β type I receptor inhibitor SB431542. In addition, the up-regulatory effect of TGF-β1 on Cx43 expression (mRNA and protein) was confirmed in primary cultures of human granulosa-lutein cells (P < 0.05). The small interfering RNA-mediated knockdown of ALK5, but not ALK4, abolished the TGF-β1-induced phosphorylation of SMAD2/3 and the up-regulation of Cx43. Furthermore, knockdown of SMAD2/3 or the common SMAD, SMAD4, abolished the stimulatory effects of TGF-β1 on Cx43 expression in SVOG cells. The TGF-β1-induced up-regulation of Cx43 contributed to the increase of GJIC between SVOG cells (P < 0.001).

Limitations, reasons for caution: The results of this study were generated from in vitro system and may not reflect the intra-ovarian microenvironment in vivo.

Wider implications of the findings: Our studies represent the first comprehensive research of molecular mechanisms of TGF-β1 in the regulation of Cx43 expression and GJIC in human granulosa cells and demonstrate that TGF-β1 may play a crucial role in the local modulation of cell-cell communication. Deepening our understanding of the molecular determinants will offer important insights into ovarian physiology and lead to the development of potential therapeutic methods for fertility regulation.

Study funding/competing interests: This research was supported by an operating grant from the Canadian Institutes of Health Research to P.C.K.L. There are no conflicts of interest to declare.

Trial registration number: NA.

Keywords: ALK5; SMAD; connexin43; human granulosa cells; transforming growth factor-β1.

Figure 1

Transforming growth factor-β1 (TGF-β1) up-regulates connexin43 (Cx43) in human granulosa cells. (A) SVOG cells were treated for 6 h with different concentrations (0.1, 1 or 10 ng/ml) of TGF-β1 and Cx43 mRNA levels were examined by RT-qPCR. (B) SVOG cells were treated for 12 h with different concentrations (0.1, 1 or 10 ng/ml) of TGF-β1 and Cx43 protein levels were examined by western blot. (C) SVOG cells were treated with 5 ng/ml TGF-β1 for 3, 6, 12 or 24 h and Cx43 mRNA levels were examined by RT-qPCR. (D) SVOG cells were treated with 5 ng/ml TGF-β1 for 6, 12 or 24 h and Cx43 protein levels were examined by western blot. (E) Primary human granulosa-lutein (hGL) cells were treated for 6 h with different concentrations (0.1, 1 or 10 ng/ml) of TGF-β1 and Cx43 mRNA levels were examined by RT-qPCR. (F) Primary hGL cells were treated for 12 h with different concentrations (0.1, 1 or 10 ng/ml) of TGF-β1 and Cx43 protein levels were examined by western blot. All western blot data are normalized to α-tubulin (tubulin). The results are expressed as the mean ± SEM of at least three independent experiments. Values marked by different letters are significantly different (P < 0.05, one-way analysis of variance (ANOVA)). Ctrl, control.

Figure 2

TGF-β1 does not affect the mRNA or protein levels of connexin37 (Cx37) in SVOG cells. (A and C) Cells were treated for 24 h with vehicle control (Ctrl) or different concentrations (0.1, 1 or 10 ng/ml) of TGF-β1 and the mRNA (A) or protein (C) levels of Cx37 were examined by RT-qPCR or western blot. (B and D) Cells were treated with 5 ng/ml TGF-β1 for 3, 6, 12 or 24 h and Cx37 mRNA (B) or protein (D) levels were examined by RT-qPCR or western blot. The results are expressed as the mean ± SEM of at least three independent experiments.

Figure 3

Effects of a TGF-β/activin type I receptor inhibitor (SB431542) on TGF-β1-induced phosphorylation of SMAD2/3 and up-regulation of Cx43 in SVOG cells. (A and B) Cells were treated with TGF-β1 (5 ng/ml) for 30 or 60 min and levels of phosphorylated Smad- and Mad-related protein (SMAD)2 (A) and SMAD3 (B) were examined by western blot (quantified data are normalized to total SMAD2 or total SMAD3, as appropriate). (C and D) Cells were treated for 60 min with TGF-β1 (5 ng/ml) in the absence (DMSO) or presence of 5 μM SB431542 (an inhibitor of TGF-β1 type I receptor) and phosphorylated SMAD2 (C) and SMAD3 (D) levels were examined by western blot. (E) Cells were treated for 6 h with TGF-β1 (5 ng/ml) in the absence (DMSO) or presence of 5 μM SB431542 and Cx43 mRNA levels were examined by RT-qPCR. (F) Cells were treated for 12 h with TGF-β1 (5 ng/ml) in the absence (DMSO) or presence of 5 μM SB431542 and Cx43 protein levels were examined by western blot. The results are expressed as the mean ± SEM of at least three independent experiments. Values marked by different letters are significantly different (P < 0.0001, one-way ANOVA).

Figure 4

Activin receptor-like kinase (ALK)5 mediates the stimulatory effects of TGF-β1 on SMA2/3 phosphorylation and Cx43 expression in SVOG cells. (A) SVOG cells were transfected for 24 or 48 h with 25 nM control siRNA (siCtrl), ALK4 siRNA (siALK4) or ALK5 siRNA (siALK5). The specificity and efficiency of knockdown for each siRNA were evaluated by RT-qPCR. (B and C) Thereafter, the effects of TGF-β1 (5 ng/ml) on SMA2/3 phosphorylation (60 min) were examined in cells previously transfected for 48 h with 25 nM siCtrl, siALK4 or siALK5. (D, E, F and G) The effects of TGF-β1 (5 ng/ml) on Cx43 mRNA levels (6 h) or Cx43 protein levels (12 h) were examined in cells previously transfected for 48 h with 25 nM siCtrl or siALK4 (D and E) as well as 25 nM siCtrl or siALK5 (F and G). The results are expressed as the mean ± SEM of at least three independent experiments. Values marked by different letters are significantly different (P < 0.001, one-way ANOVA).

Figure 5

SMAD2/3-SMAD4 signaling is required for TGF-β1-induced up-regulation of Cx43 in SVOG cells. (A) SVOG cells were transfected for 24 or 48 h with 25 nM siCtrl, SMAD2 siRNA (siSMAD2), SMAD3 siRNA (siSMAD3) or SMAD4 siRNA (siSMAD4). The specificity and efficiency of knockdown for each siRNA were evaluated by RT-qPCR. (B and C) Following transfection for 48 h with 25 nM of siCtrl, siSMAD2, siSMAD3 or combined siSMAD2 and siSMAD3 (siSMAD2+3), SVOG cells were treated with TGF-β1 (5 ng/ml) and Cx43 mRNA levels (6 h) and Cx43 protein levels (12 h) were examined by RT-qPCR and western blot. (D and E) Following transfection for 48 h with 25 nM of siCtrl or siSMAD4, SVOG cells were treated with TGF-β1 (5 ng/ml) and Cx43 mRNA levels (6 h) and Cx43 protein levels (12 h) were examined by RT-qPCR and western blot, respectively. The results are expressed as the mean ± SEM of at least three independent experiments. Values marked by different letters are significantly different (P < 0.001, one-way ANOVA).

Figure 6

TGF-β1 increases gap junction intercellular communication activities (GJIC) in SVOG cells. Fully confluent SVOG cells were treated with 5 ng/ml TGF-β1 for 24 h in the presence or absence of 5 μM SB431542 (upper panels.). The GJIC were measured by monitoring the transfer of Lucifer yellow fluorescent dye between cells, and the images were captured utilizing a fluorescence microscope. Lucifer yellow fluorescent dye entered the cells at the border of the scratch (straight white line) and reached cells away from the scratch (red arrow). Scale bar represents 25 μm. Magnification, ×200. The results are expressed as the mean ± SEM of at least three independent experiments. Values marked by different letters are significantly different (P < 0.001, one-way ANOVA).