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Lactation failure in Src knockout mice is due to impaired secretory activation.

Watkin H, Richert MM, Lewis A, Terrell K, McManaman JP, Anderson SM - BMC Dev. Biol. (2008)

Bottom Line: Failed secretory activation results in precocious involution in the mammary glands of Src-/- even when pups were suckling.Involution was accelerated following pup withdrawal perhaps as a result of incomplete secretory activation.Src appears to be required for increased expression of the prolactin receptor and successful downstream signaling, and alveolar cell organization.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology, University of Colorado Health Sciences Center, Research Complex I, South Tower, Mail Stop 8104, 12801 East 17th Avenue, Aurora, CO 80045, USA. harriet.watkin@uchsc.edu

ABSTRACT

Background: Mammary gland development culminates in lactation and is orchestrated by numerous stimuli and signaling pathways. The Src family of nonreceptor tyrosine kinases plays a pivotal role in cell signaling. In order to determine if Src plays a role in mammary gland development we have examined mammary gland development and function during pregnancy and lactation in mice in which expression of Src has been eliminated.

Results: We have characterized a lactation defect in the Src-/- mice which results in the death of over 80% of the litters nursed by Src-/- dams. Mammary gland development during pregnancy appears normal in these mice; however secretory activation does not seem to occur. Serum prolactin levels are normal in Src-/- mice compared to wildtype controls. Expression of the prolactin receptor at both the RNA and protein level was decreased in Src-/- mice following the transition from pregnancy to lactation, as was phosphorylation of STAT5 and expression of milk protein genes. These results suggest that secretory activation, which occurs following parturition, does not occur completely in Src-/- mice. Failed secretory activation results in precocious involution in the mammary glands of Src-/- even when pups were suckling. Involution was accelerated following pup withdrawal perhaps as a result of incomplete secretory activation. In vitro differentiation of mammary epithelial cells from Src-/- mice resulted in diminished production of milk proteins compared to the amount of milk proteins produced by Src+/+ cells, indicating a direct role for Src in regulating the transcription/translation of milk protein genes in mammary epithelial cells.

Conclusion: Src is an essential signaling modulator in mammary gland development as Src-/- mice exhibit a block in secretory activation that results in lactation failure and precocious involution. Src appears to be required for increased expression of the prolactin receptor and successful downstream signaling, and alveolar cell organization.

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Model of the pathways required for secretory activation and lactation that are perturbed in Src-/- mice. A) PRLR EXPRESSION. In normal mice PRLR expression increases at parturition, this is absent in the Src-/- mice and they have decreased levels of both PRLR message and protein. B) INTEGRIN CROSSTALK. Integrin-mediated adhesion is essential for STAT5 activation downstream of PRLR signaling and Src may be a required mediator for the β 1 integrin-induced crosstalk. The Src-/- mice have diminished levels of STAT5 phosphorylation. C) POLARITY. Epithelial cell polarity is crucial for secretory activation. The mammary glands of Src-/- mice do not have organized lobuloalveolar structures and many of the epithelial cells do not show restricted apical staining suggesting that Src is required for epithelial cell polarity and organization. D) MILK PROTEIN TRANSCRIPTION. Src-/- mice have significantly less β-casein message compared to wildtype mice. This may be directly related to the decreased levels of STAT5 activation in these cells or Src may also be a required signaling molecule in the additional pathways necessary for milk protein transcription. E) MILK PROTEIN TRANSLATION. Milk protein expression is also regulated at the translational level. Src-/- mice have decreased levels of milk proteins and a possible role for Src in the post-transcriptional regulation has not been investigated.
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Figure 11: Model of the pathways required for secretory activation and lactation that are perturbed in Src-/- mice. A) PRLR EXPRESSION. In normal mice PRLR expression increases at parturition, this is absent in the Src-/- mice and they have decreased levels of both PRLR message and protein. B) INTEGRIN CROSSTALK. Integrin-mediated adhesion is essential for STAT5 activation downstream of PRLR signaling and Src may be a required mediator for the β 1 integrin-induced crosstalk. The Src-/- mice have diminished levels of STAT5 phosphorylation. C) POLARITY. Epithelial cell polarity is crucial for secretory activation. The mammary glands of Src-/- mice do not have organized lobuloalveolar structures and many of the epithelial cells do not show restricted apical staining suggesting that Src is required for epithelial cell polarity and organization. D) MILK PROTEIN TRANSCRIPTION. Src-/- mice have significantly less β-casein message compared to wildtype mice. This may be directly related to the decreased levels of STAT5 activation in these cells or Src may also be a required signaling molecule in the additional pathways necessary for milk protein transcription. E) MILK PROTEIN TRANSLATION. Milk protein expression is also regulated at the translational level. Src-/- mice have decreased levels of milk proteins and a possible role for Src in the post-transcriptional regulation has not been investigated.

Mentions: We and others have described that Src is required for ductal elongation during puberty in Src-/- mice. Kim et al have suggested that this defect is due to diminished estrogen receptor signaling in the mammary gland [31], while we hypothesize that expression of Src is required in the stromal cell compartment (Richert et al, unpublished data). The data presented in this manuscript describe a novel role for Src in the mammary gland during secretory activation and/or lactation that was not previously anticipated by the phenotype of the Src knockout mice. We have observed that the growth of pups nursed by Src-/- dams is severely diminished and over 80% of the litters do not survive more than a few days. This phenotype is determined by the genotype of the dam and not the pups such that the growth of FVB, C57Bl6 and Src+/- pups nursed by Src-/- dams was diminished. The fact that over 90% of pups born to Src-/- mothers die within days, and that over 80% of fostered litters also die, indicates that a failed lactation is the predominant phenotype. The variation in the histology of the lactating mammary gland may reflect a physiological state in which survival of mammary epithelial cells is supported even when lactation is compromised. Analysis of the mammary gland of Src-/- mice during pregnancy and lactation reveals that large CLDs accumulate in the MECs of Src-/- mice following parturition providing evidence that secretory activation has not occurred [10]. Serum PRL levels in the Src-/- mice are normal indicating that neuronal stimulation of the hypothalamus, release of prolactin releasing factor, the response of the pituitary to this stimulus and hormonal signals from the pituitary are not affected by the absence of Src. We have demonstrated by gene expression profiling that expression of the long form of the PRLR increases at parturition in the mammary gland of normal mice [7,8], which is in agreement with other data using PCR-based approaches [42], however it appears that expression of the PRLR is diminished, although not absent, in the mammary glands of Src-/- mice (Figure 11). The decreased expression of the PRLR in the Src-/- mice can thus account for the diminished activation of the STAT5 transcription factor, which regulates expression of milk protein genes.


Lactation failure in Src knockout mice is due to impaired secretory activation.

Watkin H, Richert MM, Lewis A, Terrell K, McManaman JP, Anderson SM - BMC Dev. Biol. (2008)

Model of the pathways required for secretory activation and lactation that are perturbed in Src-/- mice. A) PRLR EXPRESSION. In normal mice PRLR expression increases at parturition, this is absent in the Src-/- mice and they have decreased levels of both PRLR message and protein. B) INTEGRIN CROSSTALK. Integrin-mediated adhesion is essential for STAT5 activation downstream of PRLR signaling and Src may be a required mediator for the β 1 integrin-induced crosstalk. The Src-/- mice have diminished levels of STAT5 phosphorylation. C) POLARITY. Epithelial cell polarity is crucial for secretory activation. The mammary glands of Src-/- mice do not have organized lobuloalveolar structures and many of the epithelial cells do not show restricted apical staining suggesting that Src is required for epithelial cell polarity and organization. D) MILK PROTEIN TRANSCRIPTION. Src-/- mice have significantly less β-casein message compared to wildtype mice. This may be directly related to the decreased levels of STAT5 activation in these cells or Src may also be a required signaling molecule in the additional pathways necessary for milk protein transcription. E) MILK PROTEIN TRANSLATION. Milk protein expression is also regulated at the translational level. Src-/- mice have decreased levels of milk proteins and a possible role for Src in the post-transcriptional regulation has not been investigated.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 11: Model of the pathways required for secretory activation and lactation that are perturbed in Src-/- mice. A) PRLR EXPRESSION. In normal mice PRLR expression increases at parturition, this is absent in the Src-/- mice and they have decreased levels of both PRLR message and protein. B) INTEGRIN CROSSTALK. Integrin-mediated adhesion is essential for STAT5 activation downstream of PRLR signaling and Src may be a required mediator for the β 1 integrin-induced crosstalk. The Src-/- mice have diminished levels of STAT5 phosphorylation. C) POLARITY. Epithelial cell polarity is crucial for secretory activation. The mammary glands of Src-/- mice do not have organized lobuloalveolar structures and many of the epithelial cells do not show restricted apical staining suggesting that Src is required for epithelial cell polarity and organization. D) MILK PROTEIN TRANSCRIPTION. Src-/- mice have significantly less β-casein message compared to wildtype mice. This may be directly related to the decreased levels of STAT5 activation in these cells or Src may also be a required signaling molecule in the additional pathways necessary for milk protein transcription. E) MILK PROTEIN TRANSLATION. Milk protein expression is also regulated at the translational level. Src-/- mice have decreased levels of milk proteins and a possible role for Src in the post-transcriptional regulation has not been investigated.
Mentions: We and others have described that Src is required for ductal elongation during puberty in Src-/- mice. Kim et al have suggested that this defect is due to diminished estrogen receptor signaling in the mammary gland [31], while we hypothesize that expression of Src is required in the stromal cell compartment (Richert et al, unpublished data). The data presented in this manuscript describe a novel role for Src in the mammary gland during secretory activation and/or lactation that was not previously anticipated by the phenotype of the Src knockout mice. We have observed that the growth of pups nursed by Src-/- dams is severely diminished and over 80% of the litters do not survive more than a few days. This phenotype is determined by the genotype of the dam and not the pups such that the growth of FVB, C57Bl6 and Src+/- pups nursed by Src-/- dams was diminished. The fact that over 90% of pups born to Src-/- mothers die within days, and that over 80% of fostered litters also die, indicates that a failed lactation is the predominant phenotype. The variation in the histology of the lactating mammary gland may reflect a physiological state in which survival of mammary epithelial cells is supported even when lactation is compromised. Analysis of the mammary gland of Src-/- mice during pregnancy and lactation reveals that large CLDs accumulate in the MECs of Src-/- mice following parturition providing evidence that secretory activation has not occurred [10]. Serum PRL levels in the Src-/- mice are normal indicating that neuronal stimulation of the hypothalamus, release of prolactin releasing factor, the response of the pituitary to this stimulus and hormonal signals from the pituitary are not affected by the absence of Src. We have demonstrated by gene expression profiling that expression of the long form of the PRLR increases at parturition in the mammary gland of normal mice [7,8], which is in agreement with other data using PCR-based approaches [42], however it appears that expression of the PRLR is diminished, although not absent, in the mammary glands of Src-/- mice (Figure 11). The decreased expression of the PRLR in the Src-/- mice can thus account for the diminished activation of the STAT5 transcription factor, which regulates expression of milk protein genes.

Bottom Line: Failed secretory activation results in precocious involution in the mammary glands of Src-/- even when pups were suckling.Involution was accelerated following pup withdrawal perhaps as a result of incomplete secretory activation.Src appears to be required for increased expression of the prolactin receptor and successful downstream signaling, and alveolar cell organization.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology, University of Colorado Health Sciences Center, Research Complex I, South Tower, Mail Stop 8104, 12801 East 17th Avenue, Aurora, CO 80045, USA. harriet.watkin@uchsc.edu

ABSTRACT

Background: Mammary gland development culminates in lactation and is orchestrated by numerous stimuli and signaling pathways. The Src family of nonreceptor tyrosine kinases plays a pivotal role in cell signaling. In order to determine if Src plays a role in mammary gland development we have examined mammary gland development and function during pregnancy and lactation in mice in which expression of Src has been eliminated.

Results: We have characterized a lactation defect in the Src-/- mice which results in the death of over 80% of the litters nursed by Src-/- dams. Mammary gland development during pregnancy appears normal in these mice; however secretory activation does not seem to occur. Serum prolactin levels are normal in Src-/- mice compared to wildtype controls. Expression of the prolactin receptor at both the RNA and protein level was decreased in Src-/- mice following the transition from pregnancy to lactation, as was phosphorylation of STAT5 and expression of milk protein genes. These results suggest that secretory activation, which occurs following parturition, does not occur completely in Src-/- mice. Failed secretory activation results in precocious involution in the mammary glands of Src-/- even when pups were suckling. Involution was accelerated following pup withdrawal perhaps as a result of incomplete secretory activation. In vitro differentiation of mammary epithelial cells from Src-/- mice resulted in diminished production of milk proteins compared to the amount of milk proteins produced by Src+/+ cells, indicating a direct role for Src in regulating the transcription/translation of milk protein genes in mammary epithelial cells.

Conclusion: Src is an essential signaling modulator in mammary gland development as Src-/- mice exhibit a block in secretory activation that results in lactation failure and precocious involution. Src appears to be required for increased expression of the prolactin receptor and successful downstream signaling, and alveolar cell organization.

Show MeSH
Related in: MedlinePlus