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Perturbing the cellular levels of steroid receptor coactivator-2 impairs murine endometrial function.

Szwarc MM, Kommagani R, Jeong JW, Wu SP, Tsai SY, Tsai MJ, O'Malley BW, DeMayo FJ, Lydon JP - PLoS ONE (2014)

Bottom Line: As pleiotropic coregulators, members of the p160/steroid receptor coactivator (SRC) family control a broad spectrum of transcriptional responses that underpin a diverse array of physiological and pathophysiological processes.Because of their potent coregulator properties, strict controls on SRC expression levels are required to maintain normal tissue functionality.This deficiency is significant since SRC involvement in many of these disorders is based on unscheduled increases in the levels (rather than the absence) of SRC expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas, United States of America.

ABSTRACT
As pleiotropic coregulators, members of the p160/steroid receptor coactivator (SRC) family control a broad spectrum of transcriptional responses that underpin a diverse array of physiological and pathophysiological processes. Because of their potent coregulator properties, strict controls on SRC expression levels are required to maintain normal tissue functionality. Accordingly, an unwarranted increase in the cellular levels of SRC members has been causally linked to the initiation and/or progression of a number of clinical disorders. Although knockout mouse models have underscored the critical non-redundant roles for each SRC member in vivo, there are surprisingly few mouse models that have been engineered to overexpress SRCs. This deficiency is significant since SRC involvement in many of these disorders is based on unscheduled increases in the levels (rather than the absence) of SRC expression. To address this deficiency, we used recent mouse technology that allows for the targeted expression of human SRC-2 in cells which express the progesterone receptor. Through cre-loxP recombination driven by the endogenous progesterone receptor promoter, a marked elevation in expression levels of human SRC-2 was achieved in endometrial cells that are positive for the progesterone receptor. As a result of this increase in coregulator expression, female mice are severely subfertile due to a dysfunctional uterus, which exhibits a hypersensitivity to estrogen exposure. Our findings strongly support the proposal from clinical observations that increased levels of SRC-2 are causal for a number of endometrial disorders which compromise fertility. Future studies will use this mouse model to decipher the molecular mechanisms that underpin the endometrial defect. We believe such mechanistic insight may provide new molecular descriptors for diagnosis, prognosis, and/or therapy in the clinical management of female infertility.

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The SRC-2:OE bigenic mouse expresses high levels of human SRC-2 in the endometrium.(A) The SRC-2:OE mouse was generated by crossing our PRcre/+ mouse [46] with the SRC-2LSL mouse (Fig. 2); the SRC-2:OE mouse is heterozygous for the PRcre and SRC-2LSL alleles. The cre/loxP genetic design selectively targets hSRC-2 expression at high levels in PR positive cells of the SRC-2:OE mouse. (B) Transcript levels of endometrial hSRC-2 in the SRC-2:OE mouse as compared to the SRC-2LSL monogenic control are shown (*p<0.05). (C) Western analysis shows high levels of SRC-2 protein expression in the SRC-2:OE endometrium as compared to the SRC-2LSL control; each lane represents pooled samples from five mice. The immunoblot also shows specific expression of the myc and Flag epitope tags in the SRC-2:OE lanes; β-actin is a loading control. (D) Immunohistochemical staining for SRC-2 and the myc-tag reveals that the hSRC-2 transgene is expressed in the luminal epithelium (LE) and glandular epithelium (GE) as well as in the stromal (S) compartment of the SRC-2:OE endometrium. Scale bar applies to all panels.
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pone-0098664-g003: The SRC-2:OE bigenic mouse expresses high levels of human SRC-2 in the endometrium.(A) The SRC-2:OE mouse was generated by crossing our PRcre/+ mouse [46] with the SRC-2LSL mouse (Fig. 2); the SRC-2:OE mouse is heterozygous for the PRcre and SRC-2LSL alleles. The cre/loxP genetic design selectively targets hSRC-2 expression at high levels in PR positive cells of the SRC-2:OE mouse. (B) Transcript levels of endometrial hSRC-2 in the SRC-2:OE mouse as compared to the SRC-2LSL monogenic control are shown (*p<0.05). (C) Western analysis shows high levels of SRC-2 protein expression in the SRC-2:OE endometrium as compared to the SRC-2LSL control; each lane represents pooled samples from five mice. The immunoblot also shows specific expression of the myc and Flag epitope tags in the SRC-2:OE lanes; β-actin is a loading control. (D) Immunohistochemical staining for SRC-2 and the myc-tag reveals that the hSRC-2 transgene is expressed in the luminal epithelium (LE) and glandular epithelium (GE) as well as in the stromal (S) compartment of the SRC-2:OE endometrium. Scale bar applies to all panels.

Mentions: As a first step toward generating a mouse model in which human SRC-2 is expressed at elevated levels in the uterus, a mouse harboring the hSRC-2LSL minigene at the ROSA26 locus was generated from targeted mouse embryonic stem cells using a homologous recombination strategy detailed in Fig. 2A. With a targeting efficiency of 42% (40/96 total ES cells screened; (Fig. 2B)), at least three targeted ES cell clones were used to successfully generate the SRC-2LSL mouse through the germline (Fig. 2C and D). To generate the SRC-2:OE mouse, our PRcre/+ mouse [46] was crossed with the hSRC-2LSL mouse line to create the PRcre/+/hSRC-2LSL or SRC-2:OE bigenic (Fig. 3A). Through cre-mediated excision, the SRC-2:OE bigenic is engineered to express high levels of hSRC-2 (driven by the CAGGS promoter) selectively in cells that express the PR. Quantitative real-time PCR clearly shows that significantly elevated levels of hSRC-2 transcripts are generated in the endometrium of the SRC-2:OE mouse as compared to the endometrium of the SRC-2LSL monogenic sibling (Fig. 3B). Importantly, transcript levels of endogenous SRC-1, -2, and -3 are not altered as a consequence of introducing elevated levels of hSRC-2 in the SRC-2:OE endometrium (Fig. S1). Similarly, elevated levels of hSRC-2 protein are expressed in the endometrium of the SRC-2:OE mouse as evidenced by western blot analysis using antibodies against hSRC-2 (which also cross reacts with endogenous mouse SRC-2) and to the epitope tags encoding Myc and Flag (Fig. 3C). Using antibodies to SRC-2 and the Myc epitope tag, immunohistochemistry clearly demonstrates an elevated level of expression for transgene-derived hSRC-2 in the luminal and glandular epithelium as well as in the stromal compartment of the SRC-2:OE endometrium (Fig. 3D). Importantly, despite expressing elevated levels of hSRC-2, the overall health and body weight of the SRC-2:OE mouse is similar to its SRC-2LSL sibling (data not shown).


Perturbing the cellular levels of steroid receptor coactivator-2 impairs murine endometrial function.

Szwarc MM, Kommagani R, Jeong JW, Wu SP, Tsai SY, Tsai MJ, O'Malley BW, DeMayo FJ, Lydon JP - PLoS ONE (2014)

The SRC-2:OE bigenic mouse expresses high levels of human SRC-2 in the endometrium.(A) The SRC-2:OE mouse was generated by crossing our PRcre/+ mouse [46] with the SRC-2LSL mouse (Fig. 2); the SRC-2:OE mouse is heterozygous for the PRcre and SRC-2LSL alleles. The cre/loxP genetic design selectively targets hSRC-2 expression at high levels in PR positive cells of the SRC-2:OE mouse. (B) Transcript levels of endometrial hSRC-2 in the SRC-2:OE mouse as compared to the SRC-2LSL monogenic control are shown (*p<0.05). (C) Western analysis shows high levels of SRC-2 protein expression in the SRC-2:OE endometrium as compared to the SRC-2LSL control; each lane represents pooled samples from five mice. The immunoblot also shows specific expression of the myc and Flag epitope tags in the SRC-2:OE lanes; β-actin is a loading control. (D) Immunohistochemical staining for SRC-2 and the myc-tag reveals that the hSRC-2 transgene is expressed in the luminal epithelium (LE) and glandular epithelium (GE) as well as in the stromal (S) compartment of the SRC-2:OE endometrium. Scale bar applies to all panels.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4048228&req=5

pone-0098664-g003: The SRC-2:OE bigenic mouse expresses high levels of human SRC-2 in the endometrium.(A) The SRC-2:OE mouse was generated by crossing our PRcre/+ mouse [46] with the SRC-2LSL mouse (Fig. 2); the SRC-2:OE mouse is heterozygous for the PRcre and SRC-2LSL alleles. The cre/loxP genetic design selectively targets hSRC-2 expression at high levels in PR positive cells of the SRC-2:OE mouse. (B) Transcript levels of endometrial hSRC-2 in the SRC-2:OE mouse as compared to the SRC-2LSL monogenic control are shown (*p<0.05). (C) Western analysis shows high levels of SRC-2 protein expression in the SRC-2:OE endometrium as compared to the SRC-2LSL control; each lane represents pooled samples from five mice. The immunoblot also shows specific expression of the myc and Flag epitope tags in the SRC-2:OE lanes; β-actin is a loading control. (D) Immunohistochemical staining for SRC-2 and the myc-tag reveals that the hSRC-2 transgene is expressed in the luminal epithelium (LE) and glandular epithelium (GE) as well as in the stromal (S) compartment of the SRC-2:OE endometrium. Scale bar applies to all panels.
Mentions: As a first step toward generating a mouse model in which human SRC-2 is expressed at elevated levels in the uterus, a mouse harboring the hSRC-2LSL minigene at the ROSA26 locus was generated from targeted mouse embryonic stem cells using a homologous recombination strategy detailed in Fig. 2A. With a targeting efficiency of 42% (40/96 total ES cells screened; (Fig. 2B)), at least three targeted ES cell clones were used to successfully generate the SRC-2LSL mouse through the germline (Fig. 2C and D). To generate the SRC-2:OE mouse, our PRcre/+ mouse [46] was crossed with the hSRC-2LSL mouse line to create the PRcre/+/hSRC-2LSL or SRC-2:OE bigenic (Fig. 3A). Through cre-mediated excision, the SRC-2:OE bigenic is engineered to express high levels of hSRC-2 (driven by the CAGGS promoter) selectively in cells that express the PR. Quantitative real-time PCR clearly shows that significantly elevated levels of hSRC-2 transcripts are generated in the endometrium of the SRC-2:OE mouse as compared to the endometrium of the SRC-2LSL monogenic sibling (Fig. 3B). Importantly, transcript levels of endogenous SRC-1, -2, and -3 are not altered as a consequence of introducing elevated levels of hSRC-2 in the SRC-2:OE endometrium (Fig. S1). Similarly, elevated levels of hSRC-2 protein are expressed in the endometrium of the SRC-2:OE mouse as evidenced by western blot analysis using antibodies against hSRC-2 (which also cross reacts with endogenous mouse SRC-2) and to the epitope tags encoding Myc and Flag (Fig. 3C). Using antibodies to SRC-2 and the Myc epitope tag, immunohistochemistry clearly demonstrates an elevated level of expression for transgene-derived hSRC-2 in the luminal and glandular epithelium as well as in the stromal compartment of the SRC-2:OE endometrium (Fig. 3D). Importantly, despite expressing elevated levels of hSRC-2, the overall health and body weight of the SRC-2:OE mouse is similar to its SRC-2LSL sibling (data not shown).

Bottom Line: As pleiotropic coregulators, members of the p160/steroid receptor coactivator (SRC) family control a broad spectrum of transcriptional responses that underpin a diverse array of physiological and pathophysiological processes.Because of their potent coregulator properties, strict controls on SRC expression levels are required to maintain normal tissue functionality.This deficiency is significant since SRC involvement in many of these disorders is based on unscheduled increases in the levels (rather than the absence) of SRC expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas, United States of America.

ABSTRACT
As pleiotropic coregulators, members of the p160/steroid receptor coactivator (SRC) family control a broad spectrum of transcriptional responses that underpin a diverse array of physiological and pathophysiological processes. Because of their potent coregulator properties, strict controls on SRC expression levels are required to maintain normal tissue functionality. Accordingly, an unwarranted increase in the cellular levels of SRC members has been causally linked to the initiation and/or progression of a number of clinical disorders. Although knockout mouse models have underscored the critical non-redundant roles for each SRC member in vivo, there are surprisingly few mouse models that have been engineered to overexpress SRCs. This deficiency is significant since SRC involvement in many of these disorders is based on unscheduled increases in the levels (rather than the absence) of SRC expression. To address this deficiency, we used recent mouse technology that allows for the targeted expression of human SRC-2 in cells which express the progesterone receptor. Through cre-loxP recombination driven by the endogenous progesterone receptor promoter, a marked elevation in expression levels of human SRC-2 was achieved in endometrial cells that are positive for the progesterone receptor. As a result of this increase in coregulator expression, female mice are severely subfertile due to a dysfunctional uterus, which exhibits a hypersensitivity to estrogen exposure. Our findings strongly support the proposal from clinical observations that increased levels of SRC-2 are causal for a number of endometrial disorders which compromise fertility. Future studies will use this mouse model to decipher the molecular mechanisms that underpin the endometrial defect. We believe such mechanistic insight may provide new molecular descriptors for diagnosis, prognosis, and/or therapy in the clinical management of female infertility.

Show MeSH
Related in: MedlinePlus