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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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Precocious activation of STAT3 and activation of MMP2 suggest early onset of involution. A) The number 4 mammary gland was removed from Src-/- and Src+/+ mice at P18 (lanes 1–6), L2 (lanes 7–12) and L9 (lanes 13–18). Three separate mice were used per genotype and developmental stage. Protein lysates were prepared as described in the Materials and Methods sections, and immunoblotting conducted to detect phosphorylated STAT3 (tyrosine7705) (top panel), the total amount of STAT3 expression (middle panel), and the amount of ERK 1&2 as a loading control (bottom panel). B) Mammary gland lysates were prepared from Src+/+ (top panel) and Src-/- (bottom panel) mice at L9. I1, I2, I4, I6, I8, and I14. The positions of the 72 kDa molecular weight proenzyme and 62 kDa molecular weight active enzyme are indicated on the left side of the panels.
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Figure 10: Precocious activation of STAT3 and activation of MMP2 suggest early onset of involution. A) The number 4 mammary gland was removed from Src-/- and Src+/+ mice at P18 (lanes 1–6), L2 (lanes 7–12) and L9 (lanes 13–18). Three separate mice were used per genotype and developmental stage. Protein lysates were prepared as described in the Materials and Methods sections, and immunoblotting conducted to detect phosphorylated STAT3 (tyrosine7705) (top panel), the total amount of STAT3 expression (middle panel), and the amount of ERK 1&2 as a loading control (bottom panel). B) Mammary gland lysates were prepared from Src+/+ (top panel) and Src-/- (bottom panel) mice at L9. I1, I2, I4, I6, I8, and I14. The positions of the 72 kDa molecular weight proenzyme and 62 kDa molecular weight active enzyme are indicated on the left side of the panels.

Mentions: There are many biochemical markers that define mammary gland involution including activation of STAT3 [50], changes in the expression of Bcl2 family members [51-53], and activation of matrix metalloproteases [54]. We examined the expression and phosphorylation/activation of STAT3 as a marker of involution since STAT3 is activated early during involution and conditional tissue-specific deletion of STAT3 results in delayed involution [50,55]. Activation of STAT3 was detected in all three of the Src knockout mammary gland lysates isolated on day two of lactation (Figure 10A top panel, lanes 1–3), and one of the three Src-/- mice analyzed on day L9 (Figure 10A, lane 9). There was no phosphorylation/activation of STAT3 on either L2 or L9 in the Src+/+ mice, even though the total levels of STAT3 expression were comparable to those in the Src-/- mice (Figure 10A, top panel, lanes 4–6 and middle panel, compare lanes 4–6 to lanes 1–3). These data provide additional evidence for precocious involution occurring in the Src-/- mice, perhaps as a result of the failed secretory activation described above.


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)

Precocious activation of STAT3 and activation of MMP2 suggest early onset of involution. A) The number 4 mammary gland was removed from Src-/- and Src+/+ mice at P18 (lanes 1–6), L2 (lanes 7–12) and L9 (lanes 13–18). Three separate mice were used per genotype and developmental stage. Protein lysates were prepared as described in the Materials and Methods sections, and immunoblotting conducted to detect phosphorylated STAT3 (tyrosine7705) (top panel), the total amount of STAT3 expression (middle panel), and the amount of ERK 1&2 as a loading control (bottom panel). B) Mammary gland lysates were prepared from Src+/+ (top panel) and Src-/- (bottom panel) mice at L9. I1, I2, I4, I6, I8, and I14. The positions of the 72 kDa molecular weight proenzyme and 62 kDa molecular weight active enzyme are indicated on the left side of the panels.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2266720&req=5

Figure 10: Precocious activation of STAT3 and activation of MMP2 suggest early onset of involution. A) The number 4 mammary gland was removed from Src-/- and Src+/+ mice at P18 (lanes 1–6), L2 (lanes 7–12) and L9 (lanes 13–18). Three separate mice were used per genotype and developmental stage. Protein lysates were prepared as described in the Materials and Methods sections, and immunoblotting conducted to detect phosphorylated STAT3 (tyrosine7705) (top panel), the total amount of STAT3 expression (middle panel), and the amount of ERK 1&2 as a loading control (bottom panel). B) Mammary gland lysates were prepared from Src+/+ (top panel) and Src-/- (bottom panel) mice at L9. I1, I2, I4, I6, I8, and I14. The positions of the 72 kDa molecular weight proenzyme and 62 kDa molecular weight active enzyme are indicated on the left side of the panels.
Mentions: There are many biochemical markers that define mammary gland involution including activation of STAT3 [50], changes in the expression of Bcl2 family members [51-53], and activation of matrix metalloproteases [54]. We examined the expression and phosphorylation/activation of STAT3 as a marker of involution since STAT3 is activated early during involution and conditional tissue-specific deletion of STAT3 results in delayed involution [50,55]. Activation of STAT3 was detected in all three of the Src knockout mammary gland lysates isolated on day two of lactation (Figure 10A top panel, lanes 1–3), and one of the three Src-/- mice analyzed on day L9 (Figure 10A, lane 9). There was no phosphorylation/activation of STAT3 on either L2 or L9 in the Src+/+ mice, even though the total levels of STAT3 expression were comparable to those in the Src-/- mice (Figure 10A, top panel, lanes 4–6 and middle panel, compare lanes 4–6 to lanes 1–3). These data provide additional evidence for precocious involution occurring in the Src-/- mice, perhaps as a result of the failed secretory activation described above.

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