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Translocation and Action of Polypeptide Hormones within the Nucleus |
1. FUNCTION OF PRL IN RESPONSIVE TISSUES
Prolactin (PRL) was originally identified as a neuroendocrine hormone of pituitary origin. While the primary function of this hormone was initially thought to lie solely within the breast, the functional pleiotropism of this peptide with regards to reproduction, osmoregulation, and behavior was subsequently recognised. Several lines of evidence have now also demonstrated an immunoregulatory role for this peptide. Structural analysis of PRL has revealed it to be related to members of the cytokine/hemato-poietin family such as growth hormone (GH), erythropoietin, granulocyte macrophage colony stimulating factor (GM-CSF) and the interleukins 2-7 . Synthesis of PRL is not limited to the hypophysis, as numerous extra-pituitary sites of PRL expression including the decidua, breast, and T-lymphocytes have been detected. The receptor for prolactin (PRLr) is present on numerous tissues including mammary epithelia, T and B lymphocytes, and macrophages. Acting through its receptor, PRL signalling stimulates cell proliferation, survival, and cellular differentiation in a tissue- and microenvironment-dependent manner. With respect to the mammary and immune systems, these data indicate that PRL acts at the endocrine, paracrine, and autocrine levels in regulating T-lymphocyte proliferation and survival and the terminal maturation of mammary tissues. Several lines of evidence have also indicated that PRL may act as both an endocrine and autocrine/paracrine progression factor for mammary carcinoma in both rodents and human.
2. TRANSDUCTION OF THE PRL SIGNAL
2.1 CELL SURFACE RECEPTOR-ASSOCIATED SIGNALS The effects of PRL on responsive tissues are mediated by the interaction of ligand with its receptor, the PRLr. As the initial event in PRL-induced signalling, binding of PRL induces PRLr dimerization. Receptor dimerization mediates the juxtaposition of the intracytoplasmic domains of the PRLr. The intracellular (signalling) domain of the PRLr contains a region of membrane-proximal homology to other cytokine receptors, i.e. the Box 1/Variable Box/Box 2/X box, as well as a unique C-terminal tail. The box 1 and 2 motifs have been implicated in Jak2 binding, and respectively consist of hydrophobic/proline and hydrophobic/acidic residues. The box 1 motif is essential for PRLr function; its deletion abrogates PRLr function. The tyrosine residues present within the C-terminus ofthe rat PRLr may also contribute to the engagement of Stat 5 and the activation of Jak2. PRLr dimerization results in the rapid phosphorylation of the PRLr signalling domains and the activation ofPRLr-associated kinases such as Jak2 and Fyn, Shc-Grb2-Sos, Vav, and Bag-1/Bc1-2. These events induce several signalling cascades, contributing to the transactivation of PRL-responsive gene loci involved in proliferation (i.e. IRF-1, cyclin B, histone H3) and the differentiated mammary phenotype (i.e. milk proteins such as b-casein).
2.2 NUCLEAR TRANSLOCATION OF PRL
The internalisation of PRL occurs within 30 min of its addition to cells expressing the PRLr. Di-leucine motifs within the intracellular domain of the PRLr are thought to mediate the internalisation of both ligand and receptor into an endosomal/multivesicular body/lysosomal pathway , While some of this internalised hormone is degraded, an appreciable quantity can be stored by the murine T-cells for up to one week of culture. By itself (i.e. in the absence of other mitogenic hormones), PRL is weakly mitogenic to breast cancer cell cultures and non-mitogenic to cultures of murine T lymphocytes. Recent data however indicate that in both lymphocytes and mammary epithelium PRL acts as a potent survival factor in the absence of other growth factors.
PRL, however, does act as a potent co-mitogen with both IL2 (on T-cells) and epidermal growth factor (EGF; on human breast cancer cells). These data indicate that PRL is necessary (but not sufficient) for cell cycle transit into S-phase during IL2-driven T-cell proliferation; in its absence, the expression of genes necessary for S-phase entry (i.e. histone, cyclins) does not occur. When both IL2-stimulated T-cells and EGF-stimulated breast cancer cells are cultured in media containing PRL, appreciable quantities of PRL (up to 10-20% of total intracellular PRL) can be detected within the cell nucleus by biochemical, immunofluorescent, and immunogold electron microscopy approaches. The internalisation of proteins from the extracellular medium through a trans-Golgi/ER pathway into the cytosol or nucleus has been previously observed with several bacterial toxins and viral proteins, and is a process known as retrotransport. In the absence of co-mitogenic stimulation (such as supplied by IL2 or EGF), the nuclear retrotranslocation of PRL does not occur, an observation noted by other laboratories. While the nuclear retrotransport of PRL could represent an epiphenomenon of cell proliferation, we hypothesised that this event was necessary for cell cycle progression. To confirm this hypothesis, three eukaryotic expression constructs of PRL were synthesised: 1) wild-type PRL, bearing its N-terminal ER leader sequence (termed “PRL/WT”), 2) a deletion construct of PRL lacking its leader sequence (“PRL/ER-”), and 3) a chimeric construct of PRL which replaced its leader sequence with the SV40 large T nuclear translocation signal sequence (“PRL/NT+”). When these constructs were transfected into the IL2- and PRL-responsive T-cell line Nb2, the expressed proteins were found within the extracellular medium, cytoplasm, and the nucleus, respectively, and were bioactive and of the appropriate size. Only the transfectant that secreted PRL into the extracellular medium (PRL/WT) was capable of proliferation in the absence of any mitogenic stimulation. In the presence of IL2, both the PRL/WT and PRL/NT+ transfectants demonstrated markedly increased proliferation (5- 10 fold increased over either the parental of PRL/ER- lines). In the presence of exogenous neutralising anti-PRL antiserum (which blocked the action of extracellular PRL), however, significant proliferation and survival of only the PRL/NT+ transfectant was noted. These data demonstrated that nuclear PRL contributed to IL2-stimulated proliferation by providing a necessary, but not sufficient, function within the cell nucleus.
The nuclear retrotranslocation of PRL has been observed in several PRL-responsive tissues including the breast, T-lymphocytes, liver, ovary, and adrenal. Other peptide hormones such as EGF, NGF, and PDGF, insulin, FGF, and IL5 have also been observed within the nucleus after their introduction into the extracellular medium. These data would indicate that the nuclear retrotranslocation of peptide hormones is a widespread phenomenon that could regulate numerous physiologic processes.
3. MECHANISMSOFPRLRETROTRANSPORTTO THE NUCLEUS,THEROLE OFCYCLOPHILINB
Given that PRL lacks intrinsic localisation motifs or enzymatic activity, it was reasoned that its nuclear retrotransport and action was mediated by PRL-associated chaperones. To identify these binding partners, yeast two-hybrid analysis was employed with PRL as “bait”. This analysis has revealed that the peptidyl-prolyl isomerase (PPI) cyclophilin B (CypB) interacts with PRL. The cyclophilins are a family of peptidyl-prolyl isomerases (PPI) that serve as protein chaperones and mediate the immunosuppressive effects of cyclosporine (CsA). Structural motifs within N- and C-termini of CypB mediate its ER/extracellular/nuclear localisation. Indeed, CypB can be found in serum and breast milk at concentrations of 150 ng/ml. Our data demonstrate that CypB directly interacted with both PRL and GH in vitro and in vivo through the use of recombinant CypB, PRL, and GH and antibodies targeted against these proteins. This interaction was significantly enhanced by the inclusion of cyclosporine A. The exogenous addition of physiologic concentrations of CypB into the defined medium of responsive cell lines potentiated PRL- and GH-driven proliferation ten- and forty-fold, respectively. CypB by itself was non-mitogenic, nor did it potentiate the action of either interleukin-2 or -3. CypB did not alter the affinity of the PRLr for its ligand, or increase the activation of PRLr-associated Jak2. The potentiation of PRL-action by CypB, however, was accompanied by a dramatic increase in the nuclear retrotranslocation of PRL. A CypB mutant, termed CypB-NT, was generated that lacked the wild-type N-terminal nuclear localisation sequence. Although CypB-NT demonstrated levels of PRL binding and PPI activity equivalent to wild type CypB, it was incapable of mediating the nuclear retrotransport of PRL or enhancing PRL-driven proliferation. These data reveal that CypB serves as a reverse chaperone for PRL that potentiates the action of this hormone. Given that cyclophilins associate with and/or modulate the activity of both the ER transporter Sec61 and known transcription factors, the interaction between CypB and PRL may provide a direct mechanism for somatolactogenic action within the nucleus.
4. CONCLUSION
While classically viewed as signalling only from the cell surface, the action of PRL and other related peptide hormones is also directly mediated within the nucleus. Thus, both peptide and steroid hormones demonstrate analogous signalling mechanisms, i.e. immediate-early signalling from the surface, and delayed-sustained signalling f'rom within the nucleus. Given the fundamental nature of the process of hormone retrotransport, novel strategies aimed at interrupting this signalling pathway may be of significant biologic and clinical utility.
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Polypeptide Hormones