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dc.contributor.advisorWheeler, Tomas T.
dc.contributor.advisorWilkins, Dick
dc.contributor.authorBeaton, Angela
dc.date.accessioned2022-10-31T00:39:10Z
dc.date.available2022-10-31T00:39:10Z
dc.date.issued1998
dc.identifier.urihttps://hdl.handle.net/10289/15299
dc.description.abstractThe regulation of milk protein gene expression in mammary epithelial cells occurs in response to multiple hormonal signals, plus cell-matrix and cell-cell interactions. A number of signalling molecules have been implicated in this process, but the exact molecular mechanisms regulating milk protein gene expression remain to be established. The project described in this thesis focuses on investigating the de novo synthesis of proteins in response to prolactin in mammary epithelial cells, to determine the role of de novo synthesis in lactogenic signalling, and to identify proteins which are induced by prolactin that could act as mediators of lactogenic signals. Two main approaches were taken: (1) The results of proteome analyses using two-dimensional electrophoresis revealed a number of prolactin-responsive proteins in cells from the mouse mammary epithelial cell line, COMMA-D, and also lactation-associated proteins in freshly isolated mouse mammary epithelial cells. Two prolactin-induced and lactation-associated proteins, p77 and p63, were identified as glucose regulated protein 78 (grp78) and protein disulphide isomerase (PDI), and assessed as potential regulators of lactation. These proteins are reticuloplasmins and probably function in mammary epithelial cells in the processing of milk proteins for secretion. The results of this investigation have shown that grp78 and PDI were not rapidly induced in response to prolactin, thus it is unlikely that these proteins are involved in mediating lactogenic signals in response to prolactin. (2) The requirement for de novo protein synthesis for maximal prolactin-stimulated β-casein gene expression was evaluated in COMMA-D cells using the protein synthesis inhibitors cycloheximide (CHX) and anisomycin. It appears that protein synthesis is required at the level of transcription for maximal induction and maintenance of β-casein gene expression (over the first 8 h of prolactin treatment). Furthermore, this effect was shown to be very rapid and independent of the lactogenic hormones, insulin and hydrocortisone, and the extracellular matrix. To determine the molecular basis for the effect of CHX on β-casein mRNA levels, STAT5 protein levels and DNA-binding activity were analysed in COMMA-D cells cultured in the presence or absence of prolactin and/or CHX. The results of Western analyses showed that STAT5 protein levels were not significantly altered in the presence or absence of (prolactin and/ or) CHX. A DNA probe, denoted STAT5(30), derived from the rat β-casein promoter sequence (-104 to -75), which contains a high affinity STAT5 consensus sequence was used for EMSA. CHX treatment stimulated the induction of a DNA-binding activity of distinct mobility to STAT5. This protein-DNA complex was shown to contain the p50 subunit of NF-κB. Detailed analysis revealed that CHX-stimulated NF-κB bound to a putative κB half-site on the STAT5(30) probe (GGAATT), which overlaps the high-affinity STAT5 DNA-binding site (TTCTTGGAA). Therefore, the effect of CHX appears to be mediated by inhibition of IκB synthesis, leading to the activation of NF-κB. Since the κB site overlaps the STAT5 binding site, it is possible that NF-κB competes for binding to the β-casein promoter and sterically hinders STAT5 binding and transactivation of β-casein transcription. It is conceivable that competition between NF-κB and STAT5 is functionally significant if NF-κB is activated in response to physiological signals in the mammary gland. This remains to be demonstrated.
dc.format.mimetypeapplication/pdf
dc.language.isoen
dc.publisherThe University of Waikato
dc.rightsAll items in Research Commons are provided for private study and research purposes and are protected by copyright with all rights reserved unless otherwise indicated.
dc.titleProlactin-induced proteins and dependence on protein synthesis for β-casein expression in mouse mammary cells
dc.typeThesis
thesis.degree.grantorThe University of Waikato
thesis.degree.levelDoctoral
thesis.degree.nameDoctor of Philosophy (PhD)
dc.date.updated2022-10-31T00:35:37Z
pubs.place-of-publicationHamilton, New Zealanden_NZ


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