The role of Niemann-Pick type C2 in zebrafish embryonic development.

Niemann-Pick disease type C (NPC) is a rare, fatal, neurodegenerative lysosomal disease caused by mutations of either NPC1 or NPC2. NPC2 is a soluble lysosomal protein that functions in coordination with NPC1 to efflux cholesterol from the lysosomal compartment. Mutations of either gene result in the accumulation of unesterified cholesterol and other lipids in the late endosome/lysosome, and reduction of cellular cholesterol bioavailability. Zygotic null npc2m/m zebrafish showed significant unesterified cholesterol accumulation at larval stages, a reduction in body size, and motor and balance defects in adulthood. However, the phenotype at embryonic stages was milder than expected, suggesting a possible role of maternal Npc2 in embryonic development. Maternal-zygotic npc2m/m zebrafish exhibited significant developmental defects, including defective otic vesicle development/absent otoliths, abnormal head/brain development, curved/twisted body axes and no circulating blood cells, and died by 72 hpf. RNA-seq analysis conducted on 30 hpf npc2+/m and MZnpc2m/m embryos revealed a significant reduction in the expression of notch3 and other downstream genes in the Notch signaling pathway, suggesting that impaired Notch3 signaling underlies aspects of the developmental defects observed in MZnpc2m/m zebrafish.

Gonadotropin-regulated testicular RNA helicase (GRTH/DDX25), a negative regulator of luteinizing/chorionic gonadotropin hormone-induced steroidogenesis in Leydig cells: central role of steroidogenic acute regulatory protein (StAR).

Gonadotropin-regulated testicular RNA helicase (GRTH/DDX25) is a testis-specific gonadotropin-regulated RNA helicase that is present in Leydig cells (LCs) and germ cells and is essential for spermatid development and completion of spermatogenesis. Normal basal levels of testosterone in serum and LCs were observed in GRTH null (GRTH(-/-)) mice. However, testosterone production was enhanced in LCs of GRTH(-/-) mice compared with WT mice by both in vivo and in vitro human chorionic gonadotropin stimulation. LCs of GRTH(-/-) mice had swollen mitochondria with a significantly increased cholesterol content in the inner mitochondrial membrane. Basal protein levels of SREBP2, HMG-CoA reductase, and steroidogenic acute regulatory protein (StAR; a protein that transports cholesterol to the inner mitochondrial membrane) were markedly increased in LCs of GRTH(-/-) mice compared with WT mice. Gonadotropin stimulation caused an increase in StAR mRNA levels and protein expression in GRTH(-/-) mice versus WT mice, with no further increase in SREBP2 and down-regulation of HMG-CoA reductase protein. The half-life of StAR mRNA was significantly increased in GRTH(-/-) mice. Moreover, association of StAR mRNA with GRTH protein was observed in WT mice. Human chorionic gonadotropin increased GRTH gene expression and its associated StAR protein at cytoplasmic sites. Taken together, these findings indicate that, through its negative role in StAR message stability, GRTH regulates cholesterol availability at the mitochondrial level. The finding of an inhibitory action of GRTH associated with gonadotropin-mediated steroidogenesis has provided insights into a novel negative autocrine molecular control mechanism of this helicase in the regulation of steroid production in the male.

Testis-specific miRNA-469 up-regulated in gonadotropin-regulated testicular RNA helicase (GRTH/DDX25)-null mice silences transition protein 2 and protamine 2 messages at sites within coding region: implications of its role in germ cell development.

Gonadotropin-regulated testicular RNA helicase (GRTH/DDX25), a testis-specific member of the DEAD-box family, is an essential post-transcriptional regulator of spermatogenesis. Failure of expression of Transition protein 2 (TP2) and Protamine 2 (Prm2) proteins (chromatin remodelers, essential for spermatid elongation and completion of spermatogenesis) with preservation of their mRNA expression was observed in GRTH-null mice (azoospermic due to failure of spermatids to elongate). These were identified as target genes for the testis-specific miR-469, which is increased in the GRTH-null mice. Further analysis demonstrated that miR-469 repressed TP2 and Prm2 protein expression at the translation level with minor effect on mRNA degradation, through binding to the coding regions of TP2 and Prm2 mRNAs. The corresponding primary-microRNAs and the expression levels of Drosha and DGCR8 (both mRNA and protein) were increased significantly in the GRTH-null mice. miR-469 silencing of TP2 and Prm2 mRNA in pachytene spermatocytes and round spermatids is essential for their timely translation at later times of spermiogenesis, which is critical to attain mature sperm. Collectively, these studies indicate that GRTH, a multifunctional RNA helicase, acts as a negative regulator of miRNA-469 biogenesis and consequently their function during spermatogenesis.

Relevance of gonadotropin-regulated testicular RNA helicase (GRTH/DDX25) in the structural integrity of the chromatoid body during spermatogenesis.

Gonadotropin-regulated testicular RNA helicase (GRTH/DDX25), a multifunctional protein and a component of ribonucleoprotein complexes, is essential for the completion of spermatogenesis. We investigated the nuclear/cytoplasmic shuttling of GRTH in germ cells and its impact on the chromatoid body (CB)-a perinuclear organelle viewed as a storage/processing site of mRNAs. GRTH resides in the nucleus, cytoplasm and CB of round spermatids. Treatment of these cells with inhibitors of nuclear export or RNA synthesis caused nuclear retention of GRTH and its absence in the cytoplasm and CB. The nuclear levels of GRTH bound RNA messages were significantly enhanced and major reduction was observed in the cytoplasm. This indicated GRTH main transport function of mRNAs to the cytoplasm and CB. MVH, a germ cell helicase, and MIWI, a component of the RNA-induced-silencing complex (RISC), confined to the CB/cytoplasm, were absent in the CB and accumulated in the cytoplasm upon treatment. This also occurred in spermatids of GRTH-KO mice. The CB changed from lobular-filamentous to a small condensed structure after treatment resembling the CB of GRTH-KO. No interaction of GRTH with MVH or RISC members in both protein and RNA were observed. Besides of participating in the transport of messages of relevant spermatogenic genes, GRTH was found to transport its own message to cytoplasmic sites. Our studies suggest that GRTH through its export/transport function as a component of mRNP is essential to govern the CB structure in spermatids and to maintain systems that may participate in mRNA storage and their processing during spermatogenesis.

The X-linked acrogigantism-associated gene gpr101 is a regulator of early embryonic development and growth in zebrafish.

We recently described X-linked acrogigantism (X-LAG), a condition of early childhood-onset pituitary gigantism associated with microduplications of the GPR101 receptor. The expression of GPR101 in hyperplastic pituitary regions and tumors in X-LAG patients, and GPR101's normally transient pituitary expression during fetal development, suggest a role in the regulation of growth. Nevertheless, little is still known about GPR101's physiological functions, especially during development. By using zebrafish models, we investigated the role of gpr101 during embryonic development and somatic growth. Transient ectopic gpr101 expression perturbed the embryonic body plan but did not affect growth. Loss of gpr101 led to a significant reduction in body size that was even more pronounced in the absence of maternal transcripts, as well as subfertility. These changes were accompanied by gastrulation and hypothalamic defects. In conclusion, both gpr101 loss- and gain-of-function affect, in different ways, fertility, embryonic patterning, growth and brain development.

Gonadotropin-regulated testicular RNA helicase (GRTH/DDX25) gene: cell-specific expression and transcriptional regulation by androgen in transgenic mouse testis.

Gonadotropin-regulated testicular RNA helicase is a multifunctional enzyme present in both Leydig and germ cells that is essential for the progress of spermatogenesis. GRTH gene expression is transcriptionally upregulated by human chorionic gonadotropin (hCG) via second messenger (cAMP) and androgen in Leydig cells. The regulatory region(s) in the GRTH gene that is/are required for its cell-specific expression in the testis and hCG/androgen dependent expression were investigated in transgenic mice carrying sequential deletions of 5' flanking sequences of the GRTH gene. GFP-reporter gene expression directed by the GRTH 5' flanking sequences extending to -3.6 kb was specifically located in Leydig cells and the 205 bp minimal promoter domain was sufficient for this cell-specific expression. The 1 kb (5' to the ATG codon) transgene-directed expression was markedly increased by in vivo hCG treatment. Administration of the androgen receptor inhibitor Flutamide blocked the basal and hCG stimulated GFP expression in Leydig cells. We conclude that the expression of GRTH in testicular cells is differentially regulated by its 5' flanking sequence and that the 1 kb fragment of GRTH gene contains sequences for androgen regulation of its expression in Leydig cells.

Gonadotropin-regulated testicular helicase (GRTH/DDX25): an essential regulator of spermatogenesis.

Male germ-cell maturation is orchestrated by a cascade of temporally regulated factors. Gonadotropin-regulated testicular helicase (GRTH/DDX25), a target of gonadotropin and androgen action, is a post-transcriptional regulator of key spermatogenesis genes. Male mice lacking GRTH are sterile, with spermatogenic arrest owing to the failure of round spermatids to elongate. GRTH is a component of messenger ribonucleoprotein particles, which transport target mRNAs to the cytoplasm for storage in chromatoid bodies of spermatids; these messages are released for translation during spermatogenesis. GRTH is also found in polyribosomes, where it regulates the translation of mRNAs encoding spermatogenesis factors. The association of GRTH mutations with male infertility underlines the importance of GRTH as a central, post-transcriptional regulator of spermatogenesis.

Modeling Niemann-Pick disease type C1 in zebrafish: a robust platform for in vivo screening of candidate therapeutic compounds.

Niemann-Pick disease type C1 (NPC1) is a rare autosomal recessive lysosomal storage disease primarily caused by mutations in NPC1 NPC1 is characterized by abnormal accumulation of unesterified cholesterol and glycolipids in late endosomes and lysosomes. Common signs include neonatal jaundice, hepatosplenomegaly, cerebellar ataxia, seizures and cognitive decline. Both mouse and feline models of NPC1 mimic the disease progression in humans and have been used in preclinical studies of 2-hydroxypropyl-ß-cyclodextrin (2HPßCD; VTS-270), a drug that appeared to slow neurological progression in a Phase 1/2 clinical trial. However, there remains a need to identify additional therapeutic agents. High-throughput drug screens have been useful in identifying potential therapeutic compounds; however, current preclinical testing is time and labor intensive. Thus, development of a high-capacity in vivo platform suitable for screening candidate drugs/compounds would be valuable for compound optimization and prioritizing subsequent in vivo testing. Here, we generated and characterize two zebrafish npc1-null mutants using CRISPR/Cas9-mediated gene targeting. The npc1 mutants model both the early liver and later neurological disease phenotypes of NPC1. LysoTracker staining of npc1 mutant larvae was notable for intense staining of lateral line neuromasts, thus providing a robust in vivo screen for lysosomal storage. As a proof of principle, we were able to show that treatment of the npc1 mutant larvae with 2HPßCD significantly reduced neuromast LysoTracker staining. These data demonstrate the potential value of using this zebrafish NPC1 model for efficient and rapid in vivo optimization and screening of potential therapeutic compounds.This article has an associated First Person interview with the first author of the paper.

Polymorphism of the GRTH/DDX25 gene in normal and infertile Japanese men: a missense mutation associated with loss of GRTH phosphorylation.

The gonadotropin-regulated testicular RNA helicase (GRTH/Ddx25) is present in Leydig and germ cells of rodents, and is essential for fertility in mice. This study evaluated the incidence of GRTH/DDX25 gene mutations in a group of infertile patients with non-obstructive azoospermia (NOA), 85% with a preponderance of Sertoli cells in the seminiferous tubule and 15% with spermatogenic arrest, and compared them to a group of fertile subjects. Exonic sequences in the GRTH gene were screened using denaturing high-performance liquid chromatography of the genomic DNA from 143 NOA and 100 fertile Japanese men. A unique heterozygous missense mutation Arg(242)His in exon 8 was identified in 5.8% of Sertoli cell-only patients and in 1% of normal subjects. Although the mutant protein was efficiently expressed in COS-1 cells, only the 56 kDa nuclear/cytoplasmic non-phosphorylated species was present, whereas the 61 kDa cytosolic phosporylated species was absent. In addition, a silent mutation was identified in exon 11 in NOA subjects. The Arg(242)His missense mutation of the GRTH/DDX25 gene associated with expression of a protein with reduced basicity, and the absence of the phospho-GRTH species, could be of relevance to some of the functional aspects of the protein that impact on germ cell development and/or function.

Ligand-independent homo- and heterodimerization of human prolactin receptor variants: inhibitory action of the short forms by heterodimerization.

Prolactin (PRL) acts through the long form (LF) of the human PRL receptor (hPRLR) to cause differentiation of mammary epithelial cells through activation of the Janus kinase-2 (JAK2)/signal transducer and activator of transcription 5 (STAT5) pathway and subsequent transcriptional events. To determine whether the inhibitory action of hPRLR short forms (SFs; S1a and S1b) on PRL-induced signal transduction through the LF results from heterodimerization, we studied complex formation among variant forms of the hPRLR. 3'-Tagged fusion constructs, with activities comparable to the wild-type species, were used to investigate homodimer and heterodimer formation. The LF and both SFs of the hPRLR formed homodimers under nonreducing conditions, independently of PRL, but formed only monomers under reducing conditions. Coimmunoprecipitation of the cotransfected LF with the SFs (S1a or S1b) in transfected cells showed ligand-independent heterodimerization of individual SFs with the LF. Bioluminescence resonance energy transfer analysis demonstrated homo- and heterodimeric associations of hPRLR variants in human embryonic kidney 293 cells. Biotin-avidin immunoprecipitation analysis revealed that hPRLR forms are cell surface receptors and that SFs do not influence the steady state or half-life of the LF. Significant homo- and heterodimerization of biotinylated membrane hPRLR forms was observed. These findings indicate that homo- and heterodimers of hPRLR are constitutively present, and that the bivalent hormone acts on the preformed LF homodimer to induce the active signal transduction configuration. Although SF homodimers and their heterodimers with LF mediate JAK2 activation, the SF heterodimer partner lacks cytoplasmic sequences essential for activation of the JAK2/signal transducer and activator of transcription 5 pathway. This prevents the heterodimeric LF from mediating activation of PRL-induced genes.

Tissue-cell- and species-specific expression of gonadotropin-regulated long chain acyl-CoA synthetase (GR-LACS) in gonads, adrenal and brain. Identification of novel forms in the brain.

Gonadotropin-regulated long chain acyl-CoA synthetase (GR-LACS) is a novel hormonally regulated fatty acyl-CoA synthetase (FACS) with activity for long-chain fatty acids. The presence of this enzyme in the Leydig cells of the mature rat testis and its mode of regulation suggest that it participates in testicular steroidogenesis. This study demonstrates that GR-LACS expression is tissue, cell and species-specific. The 79 kDa GR-LACS protein is expressed in rodent gonads and brain, and only in the mouse in the adrenal cortex. In the ovary of both species it is associated with follicles undergoing atresia. It is present in the newborn and immature testis tubules and after puberty only in the Leydig cells. A distinct GR-LACS protein species of 64 kDa that was more abundant than the 79 kDa long form was found in the rat brain. Also, a minor 73 kDa form was observed in the rat brain and mouse ovary. Two novel species resulting from alternatively splicing of the GR-LACS gene were identified in a rat brain cDNA library: a short form 1 (S1) lacking exon 8 and short form 2 (S2) lacking exons 6-8. Expression studies revealed that the sizes of the S1/S2 proteins are comparable to those of the endogenous variant species. Neither S form contains FACSs activity, suggesting that exon 8 is essential for the enzymatic function. GR-LACS variants exhibit small but significant dominant negative effects on the FACS activity of the long form. GR-LACS variants may regulate the long form's activity in the brain.

Human prolactin receptor variants in breast cancer: low ratio of short forms to the long-form human prolactin receptor associated with mammary carcinoma.

Prolactin plays an essential role in the development of rodent mammary tumors and is a potent mitogen in human normal and cancerous breast tissues/cells. In this study, we have analyzed the expression of prolactin receptors, including the long receptor form (LF; stimulatory) and two novel short forms (SFs; S1a and S1b) derived from alternative splicing that are inhibitory of the activation induced by prolactin through the LF. Southern analysis of breast cancer profiling arrays revealed that 29 patients (group I) expressed an elevated LF, 10 patients (group II) showed decreased LF, and 8 patients (group III) had no change relative to the adjacent normal tissue. Their respective SF expression was increased in 21 patients of group I and generally decreased in groups II and III. However, the ratio of SF to LF was significantly decreased in 76% of the breast tumors and distributed evenly among the groups. Quantification of differential expression of prolactin receptor variants by real-time PCR in 15 pairs of human normal and tumor breast-matched tissues revealed a similar significant decrease in the ratio of SF to LF in the tumor tissue. Consistent lower ratio of SFs to LFs was confirmed in 8 of ten different breast cancer cell lines compared with normal mammary Hs578Bst and MCF10A cells. Because SFs act as dominant negative regulators of the stimulatory actions of the LF in vitro, their relatively reduced expression in cancer could cause gradations of unopposed prolactin-mediated LF stimulatory function and contribute to breast tumor development/progression.

The gonadotropin-regulated long-chain acyl CoA synthetase gene: a novel downstream Sp1/Sp3 binding element critical for transcriptional promoter activity.

The 79 kD gonadotropin-regulated testicular long chain acyl-CoA synthetase gene (GR-LACS) is a hormone-regulated member of the acyl-CoA synthetase family that is expressed abundantly in Leydig cells and to a lesser extent in germinal cells of the adult testis. GR-LACS possesses an ATP/AMP binding domain and the fatty acyl-CoA synthetase (FACS) signature motif. To gain insights into the transcriptional regulation of GR-LACS in gonadal cells, we determined the genomic organization of the gene, including the upstream flanking sequences. The mouse GR-LACS gene spans over at least 45 kb and the coding region is encoded by exons 1-14. All exon-intron junction sites correspond to the consensus splice sequence GT-AG. Exon 7 and 11 comprise the conserved ATP/AMP binding domain and the FACS signature motif, respectively. Primer extension and S1 nuclease analyses demonstrated four transcriptional start sites located at -266/-216 bp 5' to the ATG codon. The minimal promoter domain resides within -254/-217 bp 5' to ATG codon, and upstream sequences to -404 bp (-1035/-405 bp) contribute to the inhibition of transcription in the expressing mouse Leydig tumor cells. Removal of -217/-1 bp, containing a 23 nt GC rich sequence (-112/-90) with an Sp1/Sp3 binding element, within the 1st exon of this TATA-less promoter, significantly reduced GR-LACS gene transcription. Transcriptional activity was abolished by a 2 nt mutation of this element. Thus, functional analyses of this promoter domain indicate that transcription of GR-LACS gene requires an Sp1/Sp3 binding element downstream of the transcriptional start sites which is essential for basal promoter activity.

Elucidation of RNA binding regions of gonadotropin-regulated testicular RNA helicase (GRTH/DDX25) to transcripts of a chromatin remodeling protein essential for spermatogenesis.

BACKGROUND: Gonadotropin-regulated testicular RNA helicase (GRTH) is a testis-specific member of the DEAD-box family of RNA helicases present in Leydig and germ cells. It is a transport protein of mRNAs from nucleus to cytoplasmic sites and is essential for posttranscriptional regulation and completion of spermatogenesis. Transition protein 2 (Tp2), which associates with GRTH and is required for spermatid elongation, failed to express in GRTH null mice with impaired mRNA nuclear export. The present study determines GRTH binding motifs/regions that associate with Tp2 mRNA transcripts. MATERIALS AND METHODS: RNA-protein interaction was analyzed using biotin-labeled electrophoretic mobility gel shift assays (EMSA). 3'-biotin-labeled RNA (Tp2) was incubated with mGRTH protein (full length/sequential deletion of specific and conserved RNA helicase motifs of GRTH) expressed from in vitro TNT coupled reticulocyte lysate system. Binding specificity was further elucidated by mutagenesis and antibody supershift analysis. RESULTS: RNA-EMSA revealed that the 3' UTR of Tp2 RNA (127 nt from TGA) was retarded in presence of full length GRTH. Nucleotide sequences downstream of TGA of the Tp2 transcript (1-47 and 78-127 nt) are important for binding to GRTH. Sequential deletions/point mutations in GRTH revealed region(s) of conserved binding motifs of RNA helicases (Ia and V) essential for GRTH binding to Tp2 mRNA. CONCLUSIONS: Our studies provide insights into the association of Tp2 expression via binding to the conserved RNA binding motifs of GRTH protein and the basis for understanding GRTH in the regulation of the genes essential for germ cell elongation and completion of spermatogenesis.

Complex 5' genomic structure of the human prolactin receptor: multiple alternative exons 1 and promoter utilization.

Transcription of the prolactin receptor (PRLR) is under the control of multiple promoters. Following the recent demonstration of the human non-coding exon 1, hE1(N) (hE1(N1)) and the generic exon 1 hE1(3), we have identified their promoters and characterized four other novel human exons 1 (hE1(N2-5)) that are alternatively spliced to a common non-coding exon 2 in human tissues and breast cancer cells. Genomic regions containing these exons, and 5'-flanking and intronic sequences, were determined and their order was established in chromosome 5p14-13. Promoters utilized in the transcription of previously characterized PRLR exons 1 species hE1(3) (hPII) and hE1(N1) (hP(N1)) were found to employ distinct mechanisms for controlling hPRLR transcription. hPIII requires C/EBP beta and Sp1/Sp3 for basal transcriptional activity, while hP(N1) activity is conferred by domains containing an Ets element and an NR half-site. The complex promoter control system that governs transcription of the hPRLR in multiple tissues is of relevance for studies on the regulation of PRLR expression in physiological and pathological states.

Genomic organization and transcriptional analysis of gonadotropin-regulated testicular RNA helicase--GRTH/DDX25 gene.

The gonadotropin-regulated testicular RNA helicase (GRTH/DDX25) is a new member of the DEAD-box protein family. Phylogenetic analysis revealed that GRTH is distantly related to other members of the family. GRTH is transcriptionally up-regulated by gonadotropin, displays ATPase and RNA helicase activities, and participates in germ cell development. To understand the regulation of GRTH gene expression, we investigated its structural organization and aspects of basal transcriptional regulation at the promoter domain. The 20-kb mouse GRTH gene contains 12 coding exons and all but one of its conserved helicase motifs are contained within single exons. GRTH is a TATA-less gene with multiple transcriptional start sites (TSS), GC-rich sequences and a promoter located within -205/+63 bp of the gene. Sequences -852/-354 and -501/-354 bp caused 40-60% and >80% inhibition of transcription in expressing and non-expressing cells, respectively. Transcriptional activity was recovered only in expressing cells by the addition of upstream sequences (-1085/-852 bp). Sp1/Sp3 supported basal transcriptional activity in all cell types, while E-box was an activator-binding site only in non-expressing cells. These findings indicate that a differential pattern of transcriptional regulation may be involved in the control of GRTH gene expression in a cell-specific manner.

Estradiol stimulates expression of two human prolactin receptor isoforms with alternative exons-1 in T47D breast cancer cells.

Human prolactin receptor (hPRLR) expression is regulated by estradiol-17beta (E(2)) in vivo in animal tissues, and in vitro in normal human endometrial cells and in MCF7 human breast cancer cells. The objective of this study was to determine the effect of E(2) on the expression of two recently described hPRLR isoforms with distinct exons-1, hE1(3) and hE1(N1) that are transcribed from the generic hPIII promoter, also present in the rat and mouse, and the human-specific promoter hP(N1), respectively. Also, to determine the effect of estradiol on the hPIII promoter activity in cancer cells. T47D breast cancer cells were examined using quantitative competitive RT-PCR for the level of expression of two alternative non-coding exon-1 transcripts, hE1(3) and hE1(N1) following incubation with E(2) in presence or absence of the E(2) receptor antagonist ICI 182,780. The effects of estradiol were also evaluated in cells transiently transfected with constructs of hPIII promoter luciferase reporter gene. E(2) significantly increased the expression of both hPRLR mRNA transcripts, hE1(3) and hE1(N1). In transfection studies E(2) activated the hPIII promoter. This effect of estradiol was markedly inhibited by coincubation with the E(2) receptor antagonist. Our results demonstrate a stimulatory effect of estradiol on the expression of hPRLR mRNA species with alternative exons-1, hE1(3) and hE1(N1) possibly through activation of their corresponding promoters. The lack of a formal ERE in these promoters suggested that the effect of estradiol is mediated through association of the activated ER with relevant DNA binding transfactor(s). These findings support the role of E(2) in the regulation of hPRLR expression in human breast cancer cell lines.

Prolactin induces up-regulation of its cognate receptor in breast cancer cells via transcriptional activation of its generic promoter by cross-talk between ERα and STAT5.

Prolactin (PRL) serves a critical role in breast cancer progression via activation of its cognate receptor. These studies reveal up-regulation of PRLR gene expression by PRL in absence of estradiol in MCF-7 and T47D breast cancer cells. PRL/PRLR via activation of STAT5 that binds a GAS-element in the PRLR gene and the participation of ERα stimulates PRLR transcription/expression. PRL/PRLR induces phosphorylation of ERα through the JAK2/PI3K/MAPK/ERK and JAK2/HER2 activated pathways. The increased recruitment of phospho-ERα, induced by PRL to Sp1 and C/EBPß at PRLR promoter sites is essential for PRL-induced PRLR transcription. This recruitment is prevented by blockade of PRL expression using RNA interference or ERα phosphorylation using specific inhibitors of PI3K and ERK. Direct evidence is provided for local actions of PRL, independent of estradiol, in the up-regulation of PRLR transcription/expression by an activation-loop between STAT5 and the phospho-ERα/Sp1/C/EBPß complex with requisite participation of signaling mechanisms. PRL's central role in the up-regulation of PRLR maximizes the action of the endogenous hormone. This study offers mechanistically rational basis for invasiveness fueled by prolactin in refractory states to adjuvant therapies in breast cancer.