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PIKE-A is required for prolactin-mediated STAT5a activation in mammary gland development.

ABSTRACT: PI 3-kinase enhancer A (PIKE-A) is critical for the activation of Akt signalling, and has an essential function in promoting cancer cell survival. However, its physiological functions are poorly understood. Here, we show that PIKE-A directly associates with both signal transducer and activator of transcription 5a (STAT5a) and prolactin (PRL) receptor, which is essential for PRL-provoked STAT5a activation and the subsequent gene transcription. Depletion of PIKE-A in HC11 epithelial cells diminished PRL-induced STAT5 activation and cyclin D1 expression, resulting in profoundly impaired cell proliferation in vitro. To confirm the function of PIKE-A in PRL signalling in vivo, we generated PIKE knockout (PIKE-/-) mice. PIKE-/- mice displayed a severe lactation defect that was characterized by enhanced apoptosis and impaired proliferation of mammary epithelial cells. At parturition, STAT5 activation and cyclin D1 expression were substantially reduced in the mammary epithelium of PIKE-/- mice. The defective mammary gland development in PIKE-/- mice was rescued by overexpression of a mammary-specific cyclin D1 transgene. These data establish a critical function for PIKE-A in mediating PRL functions.

SUBMITTER: Chan CB

PROVIDER: S-EPMC2837170 | biostudies-literature | 2010 Mar

REPOSITORIES: biostudies-literature

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PIKE-A is required for prolactin-mediated STAT5a activation in mammary gland development.

Chan Chi-Bun CB Liu Xia X Ensslin Michael A MA Dillehay Dirck L DL Ormandy Christopher J CJ Sohn Philip P Serra Rosa R Ye Keqiang K

The EMBO journal 20100114 5

PI 3-kinase enhancer A (PIKE-A) is critical for the activation of Akt signalling, and has an essential function in promoting cancer cell survival. However, its physiological functions are poorly understood. Here, we show that PIKE-A directly associates with both signal transducer and activator of transcription 5a (STAT5a) and prolactin (PRL) receptor, which is essential for PRL-provoked STAT5a activation and the subsequent gene transcription. Depletion of PIKE-A in HC11 epithelial cells diminish ...[more]

PMID: 20075866

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Project description:P190A and p190B Rho GTPase activating proteins (GAPs) are essential genes that have distinct, but overlapping roles in the developing nervous system. Previous studies from our laboratory demonstrated that p190B is required for mammary gland morphogenesis, and we hypothesized that p190A might have a distinct role in the developing mammary gland. To test this hypothesis, we examined mammary gland development in p190A-deficient mice. P190A expression was detected by in situ hybridization in the developing E14.5day embryonic mammary bud and within the ducts, terminal end buds (TEBs), and surrounding stroma of the developing virgin mammary gland. In contrast to previous results with p190B, examination of p190A heterozygous mammary glands demonstrated that p190A deficiency disrupted TEB morphology, but did not significantly delay ductal outgrowth indicating haploinsufficiency for TEB development. To examine the effects of homozygous deletion of p190A, embryonic mammary buds were rescued by transplantation into the cleared fat pads of SCID/Beige mice. Complete loss of p190A function inhibited ductal outgrowth in comparison to wildtype transplants (51% vs. 94% fat pad filled). In addition, the transplantation take rate of p190A deficient whole gland transplants from E18.5 embryos was significantly reduced compared to wildtype transplants (31% vs. 90%, respectively). These results suggest that p190A function in both the epithelium and stroma is required for mammary gland development. Immunostaining for p63 demonstrated that the myoepithelial cell layer is disrupted in the p190A deficient glands, which may result from the defective cell adhesion between the cap and body cell layers detected in the TEBs. The number of estrogen- and progesterone receptor-positive cells, as well as the expression levels of these receptors was increased in p190A deficient outgrowths. These data suggest that p190A is required in both the epithelial and stromal compartments for ductal outgrowth and that it may play a role in mammary epithelial cell differentiation.

Project description:Prolactin receptors present in the particulate fraction of lactating pig mammary gland were solubilized by 7.5mM-3-[(3-cholamidopropyl)dimethylammonio]-1-propane-su lph onic acid (Chaps) and purified by affinity chromatography on prolactin coupled to Affi-Gel 10. Nearly 30% of the particulate receptors were solubilized by the detergent and over a 1000-fold purification from homogenates was achieved. A water-soluble fraction rich in receptors was observed during the preparation of membranes, although this fraction has not yet been purified. Prolactin binding to the receptors was a time-dependent, reversible and saturable reaction in particulate, Chaps-solubilized and purified receptors. In all forms, receptors showed the same specificity to peptide hormones. Prolactin and human growth hormone bound to the same receptors, whereas bovine growth hormone, follicle-stimulating hormone, luteinizing hormone, thyroid-stimulating hormone and insulin failed to bind. After solubilization, the dissociation constant (Kd) for prolactin was decreased 5-fold from 9.8 X 10(-11) M in the particulate receptors to 1.8 X 10(-11) M in solubilized and purified receptors, being due principally to an increase in the association rate constant from 1.0 X 10(9)M-1 X h-1 to (3.9-4.6) X 10(9)M-1 X h-1, respectively, with the dissociation rate constant remaining unchanged at (1.1-1.3) X 10(-2)h-1. Isoelectric focusing of the prolactin-receptor complex revealed two peaks, one at a pI of 5.5-5.6 and the other at 5.2-5.3. Microsomal receptors were covalently cross-linked to 125I-labelled ovine prolactin with ethylene glycol bis(succinimidyl succinate) and analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Autoradiography of the gel revealed a major subunit of Mr 28 000-35 000 and a minor one of Mr 67 000-69 000. Anti-(prolactin receptor) antibodies raised against rabbit mammary gland prolactin receptors were equally effective in inhibiting prolactin binding to particulate, solubilized and affinity-purified receptors, suggesting that purified prolactin receptors have a structure indistinguishable immunologically from particulate receptors and rabbit mammary gland prolactin receptors. The present demonstration shows that particulate prolactin receptors from a domestic animal can be solubilized and purified without losing the original properties of high affinity and binding specificity for hormones.

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PIKE-A is required for prolactin-mediated STAT5a activation in mammary gland development.

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PIKE-A is required for prolactin-mediated STAT5a activation in mammary gland development.

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