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Rac1 Regulates Endometrial Secretory Function to Control Placental Development.

Davila J, Laws MJ, Kannan A, Li Q, Taylor RN, Bagchi MK, Bagchi IC - PLoS Genet. (2015)

Bottom Line: Ablation of Rac1 did not affect the formation of the decidua but led to fetal loss in mid gestation accompanied by extensive hemorrhage.Consequently, the Rac1- decidual cells failed to secrete vascular endothelial growth factor A, which is a critical regulator of decidual angiogenesis, and insulin-like growth factor binding protein 4, which regulates the bioavailability of insulin-like growth factors that promote proliferation and differentiation of trophoblast cell lineages in the ectoplacental cone.The lack of secretion of these key factors by Rac1- decidua gave rise to impaired angiogenesis and dysregulated proliferation of trophoblast cells, which in turn results in overexpansion of the trophoblast giant cell lineage and disorganized placenta development.

View Article: PubMed Central - PubMed

Affiliation: Department of Comparative Biosciences, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States of America.

ABSTRACT
During placenta development, a succession of complex molecular and cellular interactions between the maternal endometrium and the developing embryo ensures reproductive success. The precise mechanisms regulating this maternal-fetal crosstalk remain unknown. Our study revealed that the expression of Rac1, a member of the Rho family of GTPases, is markedly elevated in mouse decidua on days 7 and 8 of gestation. To investigate its function in the uterus, we created mice bearing a conditional deletion of the Rac1 gene in uterine stromal cells. Ablation of Rac1 did not affect the formation of the decidua but led to fetal loss in mid gestation accompanied by extensive hemorrhage. To gain insights into the molecular pathways affected by the loss of Rac1, we performed gene expression profiling which revealed that Rac1 signaling regulates the expression of Rab27b, another GTPase that plays a key role in targeting vesicular trafficking. Consequently, the Rac1- decidual cells failed to secrete vascular endothelial growth factor A, which is a critical regulator of decidual angiogenesis, and insulin-like growth factor binding protein 4, which regulates the bioavailability of insulin-like growth factors that promote proliferation and differentiation of trophoblast cell lineages in the ectoplacental cone. The lack of secretion of these key factors by Rac1- decidua gave rise to impaired angiogenesis and dysregulated proliferation of trophoblast cells, which in turn results in overexpansion of the trophoblast giant cell lineage and disorganized placenta development. Further experiments revealed that RAC1, the human ortholog of Rac1, regulates the secretory activity of human endometrial stromal cells during decidualization, supporting the concept that this signaling G protein plays a central and conserved role in controlling endometrial secretory function. This study provides unique insights into the molecular mechanisms regulating endometrial secretions that mediate stromal-endothelial and stromal-trophoblast crosstalk critical for placenta development and establishment of pregnancy.

No MeSH data available.


Related in: MedlinePlus

RAC1 regulates secretory function of human endometrial stromal cells.(A)RAC1 is induced in primary human endometrial stromal cells undergoing in vitro decidualization. Human endometrial stromal cells (HESCs) were subjected to differentiation in response to estrogen, progesterone, and 8-Br-cAMP as described in the Materials and Methods. The expression of RAC1 mRNA was assessed by qPCR. Data represent mean ± SEM from three separate samples and were analyzed by one-way ANOVA with Bonferroni post-test. Asterisks indicate statistically significant differences (*P < 0.05). (B & C) Inhibition of biological activity of RAC1 in HESCs inhibits RAB27B expression and VEGFA secretion in the conditioned media. HESCs were subjected to differentiation in the absence or presence of 25μM InSolution Rac1 Inhibitor II (Z62954982) for eight days. (B) qPCR was performed to monitor the expression of RAC1, RAC2, RHOA, CDC42, VEGFA, and RAB27B, N = 2–3, P< 0.001. (C & D) ELISA was performed to measure VEGFA and IGFBP1 secretion in the conditioned media. VEGFA ELISA data represent mean ± SEM from three separate samples and were analyzed by two-way ANOVA with Bonferroni post-test. Asterisks indicate statistically significant differences (*P < 0.05 and ***P < 0.001). IGFBP1 ELISA data represent mean ± SEM from two separate samples and were analyzed by t-test, P > 0.05.
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pgen.1005458.g008: RAC1 regulates secretory function of human endometrial stromal cells.(A)RAC1 is induced in primary human endometrial stromal cells undergoing in vitro decidualization. Human endometrial stromal cells (HESCs) were subjected to differentiation in response to estrogen, progesterone, and 8-Br-cAMP as described in the Materials and Methods. The expression of RAC1 mRNA was assessed by qPCR. Data represent mean ± SEM from three separate samples and were analyzed by one-way ANOVA with Bonferroni post-test. Asterisks indicate statistically significant differences (*P < 0.05). (B & C) Inhibition of biological activity of RAC1 in HESCs inhibits RAB27B expression and VEGFA secretion in the conditioned media. HESCs were subjected to differentiation in the absence or presence of 25μM InSolution Rac1 Inhibitor II (Z62954982) for eight days. (B) qPCR was performed to monitor the expression of RAC1, RAC2, RHOA, CDC42, VEGFA, and RAB27B, N = 2–3, P< 0.001. (C & D) ELISA was performed to measure VEGFA and IGFBP1 secretion in the conditioned media. VEGFA ELISA data represent mean ± SEM from three separate samples and were analyzed by two-way ANOVA with Bonferroni post-test. Asterisks indicate statistically significant differences (*P < 0.05 and ***P < 0.001). IGFBP1 ELISA data represent mean ± SEM from two separate samples and were analyzed by t-test, P > 0.05.

Mentions: We next assessed whether Rac1-mediated regulation of decidual secretory pathways is conserved in the human. To test this, undifferentiated human endometrial stromal cells (HESC) isolated from biopsies obtained from normal fertile women in the proliferative stage of the menstrual cycle were placed in culture and subjected to decidualization in vitro in response to a hormonal cocktail containing progesterone, estrogen, and 8-bromo-cAMP as described previously [43, 44]. As shown in Fig 8A, RAC1 transcripts are induced in HESC during in vitro decidualization. We next employed the Rac1 inhibitor Z62954982, which specifically blocks the activation of RAC1 [45], to investigate the role of this factor in regulating the secretory pathways in differentiating HESC. Our studies revealed that treatment of endometrial stromal cells with the RAC1-specific inhibitor did not affect the expression of transcripts corresponding to RAC1, RAC2, RHOA, CDC42, or VEGFA, but led to marked suppression in the level of RAB27B transcripts (Fig 8B), indicating that the regulation of RAB27B expression by RAC1 is conserved in decidua of mouse and woman. Most importantly, inactivation of RAC1 signaling and consequent down-regulation of RAB27B gene expression were associated with a strong reduction in the levels of VEGFA secreted in the conditioned media of decidualizing HESC compared to untreated HESC (Fig 8C). Interestingly, while IGFBP4 levels were undetectable in the conditioned medium of decidualizing HESC, we observed significant levels of IGFBP1 in their conditioned media. The secreted IGFBP1 levels, however, remained unaffected by the presence or absence of the RAC1-inhibitor by day 8 of culture (Fig 8D). These results indicated that RAC1 regulates the secretion of VEGFA, but not that of IGFBP1, by HESC during decidualization. Collectively, our results are consistent with the hypothesis that Rac1, acting via its downstream effector Rab27b, controls the secretory pathways that operate in decidual cells to regulate the secretion of key paracrine factors, such as VEGF, in both mouse and human endometrium.


Rac1 Regulates Endometrial Secretory Function to Control Placental Development.

Davila J, Laws MJ, Kannan A, Li Q, Taylor RN, Bagchi MK, Bagchi IC - PLoS Genet. (2015)

RAC1 regulates secretory function of human endometrial stromal cells.(A)RAC1 is induced in primary human endometrial stromal cells undergoing in vitro decidualization. Human endometrial stromal cells (HESCs) were subjected to differentiation in response to estrogen, progesterone, and 8-Br-cAMP as described in the Materials and Methods. The expression of RAC1 mRNA was assessed by qPCR. Data represent mean ± SEM from three separate samples and were analyzed by one-way ANOVA with Bonferroni post-test. Asterisks indicate statistically significant differences (*P < 0.05). (B & C) Inhibition of biological activity of RAC1 in HESCs inhibits RAB27B expression and VEGFA secretion in the conditioned media. HESCs were subjected to differentiation in the absence or presence of 25μM InSolution Rac1 Inhibitor II (Z62954982) for eight days. (B) qPCR was performed to monitor the expression of RAC1, RAC2, RHOA, CDC42, VEGFA, and RAB27B, N = 2–3, P< 0.001. (C & D) ELISA was performed to measure VEGFA and IGFBP1 secretion in the conditioned media. VEGFA ELISA data represent mean ± SEM from three separate samples and were analyzed by two-way ANOVA with Bonferroni post-test. Asterisks indicate statistically significant differences (*P < 0.05 and ***P < 0.001). IGFBP1 ELISA data represent mean ± SEM from two separate samples and were analyzed by t-test, P > 0.05.
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pgen.1005458.g008: RAC1 regulates secretory function of human endometrial stromal cells.(A)RAC1 is induced in primary human endometrial stromal cells undergoing in vitro decidualization. Human endometrial stromal cells (HESCs) were subjected to differentiation in response to estrogen, progesterone, and 8-Br-cAMP as described in the Materials and Methods. The expression of RAC1 mRNA was assessed by qPCR. Data represent mean ± SEM from three separate samples and were analyzed by one-way ANOVA with Bonferroni post-test. Asterisks indicate statistically significant differences (*P < 0.05). (B & C) Inhibition of biological activity of RAC1 in HESCs inhibits RAB27B expression and VEGFA secretion in the conditioned media. HESCs were subjected to differentiation in the absence or presence of 25μM InSolution Rac1 Inhibitor II (Z62954982) for eight days. (B) qPCR was performed to monitor the expression of RAC1, RAC2, RHOA, CDC42, VEGFA, and RAB27B, N = 2–3, P< 0.001. (C & D) ELISA was performed to measure VEGFA and IGFBP1 secretion in the conditioned media. VEGFA ELISA data represent mean ± SEM from three separate samples and were analyzed by two-way ANOVA with Bonferroni post-test. Asterisks indicate statistically significant differences (*P < 0.05 and ***P < 0.001). IGFBP1 ELISA data represent mean ± SEM from two separate samples and were analyzed by t-test, P > 0.05.
Mentions: We next assessed whether Rac1-mediated regulation of decidual secretory pathways is conserved in the human. To test this, undifferentiated human endometrial stromal cells (HESC) isolated from biopsies obtained from normal fertile women in the proliferative stage of the menstrual cycle were placed in culture and subjected to decidualization in vitro in response to a hormonal cocktail containing progesterone, estrogen, and 8-bromo-cAMP as described previously [43, 44]. As shown in Fig 8A, RAC1 transcripts are induced in HESC during in vitro decidualization. We next employed the Rac1 inhibitor Z62954982, which specifically blocks the activation of RAC1 [45], to investigate the role of this factor in regulating the secretory pathways in differentiating HESC. Our studies revealed that treatment of endometrial stromal cells with the RAC1-specific inhibitor did not affect the expression of transcripts corresponding to RAC1, RAC2, RHOA, CDC42, or VEGFA, but led to marked suppression in the level of RAB27B transcripts (Fig 8B), indicating that the regulation of RAB27B expression by RAC1 is conserved in decidua of mouse and woman. Most importantly, inactivation of RAC1 signaling and consequent down-regulation of RAB27B gene expression were associated with a strong reduction in the levels of VEGFA secreted in the conditioned media of decidualizing HESC compared to untreated HESC (Fig 8C). Interestingly, while IGFBP4 levels were undetectable in the conditioned medium of decidualizing HESC, we observed significant levels of IGFBP1 in their conditioned media. The secreted IGFBP1 levels, however, remained unaffected by the presence or absence of the RAC1-inhibitor by day 8 of culture (Fig 8D). These results indicated that RAC1 regulates the secretion of VEGFA, but not that of IGFBP1, by HESC during decidualization. Collectively, our results are consistent with the hypothesis that Rac1, acting via its downstream effector Rab27b, controls the secretory pathways that operate in decidual cells to regulate the secretion of key paracrine factors, such as VEGF, in both mouse and human endometrium.

Bottom Line: Ablation of Rac1 did not affect the formation of the decidua but led to fetal loss in mid gestation accompanied by extensive hemorrhage.Consequently, the Rac1- decidual cells failed to secrete vascular endothelial growth factor A, which is a critical regulator of decidual angiogenesis, and insulin-like growth factor binding protein 4, which regulates the bioavailability of insulin-like growth factors that promote proliferation and differentiation of trophoblast cell lineages in the ectoplacental cone.The lack of secretion of these key factors by Rac1- decidua gave rise to impaired angiogenesis and dysregulated proliferation of trophoblast cells, which in turn results in overexpansion of the trophoblast giant cell lineage and disorganized placenta development.

View Article: PubMed Central - PubMed

Affiliation: Department of Comparative Biosciences, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States of America.

ABSTRACT
During placenta development, a succession of complex molecular and cellular interactions between the maternal endometrium and the developing embryo ensures reproductive success. The precise mechanisms regulating this maternal-fetal crosstalk remain unknown. Our study revealed that the expression of Rac1, a member of the Rho family of GTPases, is markedly elevated in mouse decidua on days 7 and 8 of gestation. To investigate its function in the uterus, we created mice bearing a conditional deletion of the Rac1 gene in uterine stromal cells. Ablation of Rac1 did not affect the formation of the decidua but led to fetal loss in mid gestation accompanied by extensive hemorrhage. To gain insights into the molecular pathways affected by the loss of Rac1, we performed gene expression profiling which revealed that Rac1 signaling regulates the expression of Rab27b, another GTPase that plays a key role in targeting vesicular trafficking. Consequently, the Rac1- decidual cells failed to secrete vascular endothelial growth factor A, which is a critical regulator of decidual angiogenesis, and insulin-like growth factor binding protein 4, which regulates the bioavailability of insulin-like growth factors that promote proliferation and differentiation of trophoblast cell lineages in the ectoplacental cone. The lack of secretion of these key factors by Rac1- decidua gave rise to impaired angiogenesis and dysregulated proliferation of trophoblast cells, which in turn results in overexpansion of the trophoblast giant cell lineage and disorganized placenta development. Further experiments revealed that RAC1, the human ortholog of Rac1, regulates the secretory activity of human endometrial stromal cells during decidualization, supporting the concept that this signaling G protein plays a central and conserved role in controlling endometrial secretory function. This study provides unique insights into the molecular mechanisms regulating endometrial secretions that mediate stromal-endothelial and stromal-trophoblast crosstalk critical for placenta development and establishment of pregnancy.

No MeSH data available.


Related in: MedlinePlus