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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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Primary mammary epithelial cell cultures confirm that Src is required for maximal prolactin signaling. Primary mammary epithelial cells isolated from Src-/- (lanes 1–3 and 10–12), Src+/- (lanes 4–6 and 13–15) and Src+/+ (lanes 7–9 and 16–18) mice were cultured on Matrigel in growth medium for 3 days then the medium was changed to differentiation medium and the cells were cultured for a further 24 or 72 hours in the presence or absence of prolactin. Cellular proteins were extracted by lysis in NET buffer. A) Immunoblotting was performed to detect phosphorylation of Src (tyrosine416) (top panel), total levels of Src protein (middle panel) and the amount of ERK 1&2 (bottom panel) as a loading control. B) Immunoblotting was performed to detect phosphorylation of STAT5 (tyrosine694) (top panel), total levels of STAT5 (middle panel) and the amount of ERK 1&2 (bottom panel) as a loading control. C) Immunoblotting was performed to detect β-casein expression (top panel), the levels of Raf and the amount of ERK 1&2 as loading controls (middle and bottom panels).
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Figure 8: Primary mammary epithelial cell cultures confirm that Src is required for maximal prolactin signaling. Primary mammary epithelial cells isolated from Src-/- (lanes 1–3 and 10–12), Src+/- (lanes 4–6 and 13–15) and Src+/+ (lanes 7–9 and 16–18) mice were cultured on Matrigel in growth medium for 3 days then the medium was changed to differentiation medium and the cells were cultured for a further 24 or 72 hours in the presence or absence of prolactin. Cellular proteins were extracted by lysis in NET buffer. A) Immunoblotting was performed to detect phosphorylation of Src (tyrosine416) (top panel), total levels of Src protein (middle panel) and the amount of ERK 1&2 (bottom panel) as a loading control. B) Immunoblotting was performed to detect phosphorylation of STAT5 (tyrosine694) (top panel), total levels of STAT5 (middle panel) and the amount of ERK 1&2 (bottom panel) as a loading control. C) Immunoblotting was performed to detect β-casein expression (top panel), the levels of Raf and the amount of ERK 1&2 as loading controls (middle and bottom panels).

Mentions: To determine whether Src was required for PRL-induced activation of STAT5 and expression of β-casein we utilized primary mammary epithelial cells (MECs) isolated from the mammary glands of Src -/-, Src +/-, and Src +/+ mice. MECs were cultured on a laminin-rich basement membrane in the absence or presence of PRL, and protein lysates were harvested after 24 and 72 hours. The expression and activation of Src in the primary MECs isolated from each of the three genotypes was determined by western blotting. Src protein was detected in Src+/+ MECs, and a reduced level was detected in the Src+/- MECs, indicating that there was a dose-dependent relationship between the number of Src alleles and the level of Src protein (Figure 8A, middle panel, lanes 4–9 and 13–18). No Src protein was detected in MECs from Src-/- mice (Figure 8A, middle panel, lanes 1–3 and 10–12). The activation of Src was examined by anti-phospho-Src (tyrosine416) antibody and the amount of phosphorylated/activated Src was found to parallel the amount of Src and not to be dependent upon PRL stimulation (Figure 8A, top panel, lanes 4–9 and 13–18). We hypothesize that PRL-dependent Src activation could not be detected in the Src+/+ and Src+/- MECs due to the high basal levels of Src phosphorylation that may result from activation of integrin signaling stimulated by attachment to the laminin-rich basement membrane and the presence of insulin in the differentiation medium. Very little immunoreactive protein was detected with the anti-phospho-Src antibody in Src-/- MECs (Figure 8A, top panel, lanes 1–3 and 10–12), which contrasted with the results obtained in studies of whole mammary gland lysates from Src-/- mice (Figure 2).


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)

Primary mammary epithelial cell cultures confirm that Src is required for maximal prolactin signaling. Primary mammary epithelial cells isolated from Src-/- (lanes 1–3 and 10–12), Src+/- (lanes 4–6 and 13–15) and Src+/+ (lanes 7–9 and 16–18) mice were cultured on Matrigel in growth medium for 3 days then the medium was changed to differentiation medium and the cells were cultured for a further 24 or 72 hours in the presence or absence of prolactin. Cellular proteins were extracted by lysis in NET buffer. A) Immunoblotting was performed to detect phosphorylation of Src (tyrosine416) (top panel), total levels of Src protein (middle panel) and the amount of ERK 1&2 (bottom panel) as a loading control. B) Immunoblotting was performed to detect phosphorylation of STAT5 (tyrosine694) (top panel), total levels of STAT5 (middle panel) and the amount of ERK 1&2 (bottom panel) as a loading control. C) Immunoblotting was performed to detect β-casein expression (top panel), the levels of Raf and the amount of ERK 1&2 as loading controls (middle and bottom panels).
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Related In: Results  -  Collection

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Figure 8: Primary mammary epithelial cell cultures confirm that Src is required for maximal prolactin signaling. Primary mammary epithelial cells isolated from Src-/- (lanes 1–3 and 10–12), Src+/- (lanes 4–6 and 13–15) and Src+/+ (lanes 7–9 and 16–18) mice were cultured on Matrigel in growth medium for 3 days then the medium was changed to differentiation medium and the cells were cultured for a further 24 or 72 hours in the presence or absence of prolactin. Cellular proteins were extracted by lysis in NET buffer. A) Immunoblotting was performed to detect phosphorylation of Src (tyrosine416) (top panel), total levels of Src protein (middle panel) and the amount of ERK 1&2 (bottom panel) as a loading control. B) Immunoblotting was performed to detect phosphorylation of STAT5 (tyrosine694) (top panel), total levels of STAT5 (middle panel) and the amount of ERK 1&2 (bottom panel) as a loading control. C) Immunoblotting was performed to detect β-casein expression (top panel), the levels of Raf and the amount of ERK 1&2 as loading controls (middle and bottom panels).
Mentions: To determine whether Src was required for PRL-induced activation of STAT5 and expression of β-casein we utilized primary mammary epithelial cells (MECs) isolated from the mammary glands of Src -/-, Src +/-, and Src +/+ mice. MECs were cultured on a laminin-rich basement membrane in the absence or presence of PRL, and protein lysates were harvested after 24 and 72 hours. The expression and activation of Src in the primary MECs isolated from each of the three genotypes was determined by western blotting. Src protein was detected in Src+/+ MECs, and a reduced level was detected in the Src+/- MECs, indicating that there was a dose-dependent relationship between the number of Src alleles and the level of Src protein (Figure 8A, middle panel, lanes 4–9 and 13–18). No Src protein was detected in MECs from Src-/- mice (Figure 8A, middle panel, lanes 1–3 and 10–12). The activation of Src was examined by anti-phospho-Src (tyrosine416) antibody and the amount of phosphorylated/activated Src was found to parallel the amount of Src and not to be dependent upon PRL stimulation (Figure 8A, top panel, lanes 4–9 and 13–18). We hypothesize that PRL-dependent Src activation could not be detected in the Src+/+ and Src+/- MECs due to the high basal levels of Src phosphorylation that may result from activation of integrin signaling stimulated by attachment to the laminin-rich basement membrane and the presence of insulin in the differentiation medium. Very little immunoreactive protein was detected with the anti-phospho-Src antibody in Src-/- MECs (Figure 8A, top panel, lanes 1–3 and 10–12), which contrasted with the results obtained in studies of whole mammary gland lysates from Src-/- mice (Figure 2).

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