Dr Heather Lee
School of Biomedical Sciences and Pharmacy
- Phone:(02) 40420680
Dr Heather Lee completed her PhD at the Garvan Institute of Medical Research where she investigated hormonal control of mammary gland development and breast cancer. Her research focused on the transcription factor, Elf5, which plays key roles in breast development during pregnancy and acquisition of anti-oestrogen resistance in breast cancer. After discovering lineage-specific DNA methylation of the Elf5 promoter in the mammary epithelium, Heather decided to undertake post-doctoral research in the field of epigenetics.
In 2012, Heather relocated to Cambridge, UK, where she joined the laboratory of Professor Wolf Reik. Using mouse embryonic stem cells as an experimental model, Heather revealed unprecedented dynamic heterogeneity in DNA methylation. Heather also developed ground-breaking single-cell sequencing technologies for the parallel analysis of genome-wide DNA methylation and gene expression in the same single cell. This achievement has established Heather as a pioneer and world-leader in the field of single-cell epigenomics.
Heather joined the University of Newcastle in February 2017, and is establishing a research group at the Hunter Medical Research Institute. Her goal is to make meaningful contributions to cancer research by revealing intra-tumoural epigenetic heterogeneity.
Research interests and directions
Heather is fascinated by the enormous diversity of cell types present in the human body, and by the importance of this cellular heterogeneity in both development and disease. Heather’s diverse interests revolve around this central theme and include:
- Epigenetic regulation of development
- Dynamic regulation of DNA methylation and cellular plasticity in cancer
- Hetergoeneous responses to chemotherapeutics
Initial projects in the lab are investigating the heterogeneous response of cancer cell lines to DNA methyltransferase inhibitors, which are used to treat acute myeloid leukaemia. Using single-cell analysis this work will reveal the hidden complexities of drug action, with the potential of identifying candidate biomarkers or new therapeutic strategies. Future projects will apply single-cell analyses to characterise circulating tumour cells, therapy-resistant cancer cells or cancer-initiating cells in patient samples.
- Doctor of Philosophy, University of New South Wales
- Bachelor of Science (Honours), University of Sydney
- DNA methylation
- Single-cell sequencing
- English (Mother)
Fields of Research
|111201||Cancer Cell Biology||45|
|060404||Epigenetics (incl. Genome Methylation and Epigenomics)||45|
|060803||Animal Developmental and Reproductive Biology||10|
|Title||Organisation / Department|
|Lecturer||University of Newcastle
School of Biomedical Sciences and Pharmacy
|Dates||Title||Organisation / Department|
|1/07/2016 - 16/12/2016||Senior Research Scientist||The Babraham Institute
|1/03/2012 - 30/06/2016||Postdoctoral Research Scientist||The Babraham Institute
|20/04/2011 - 3/02/2012||Research Officer||Garvan Institute of Medical Research
Cancer Research Program
|13/12/2010 - 19/04/2011||Research Assistant||Garvan Institute of Medical Research
Cancer Research Program
|Year||Title / Rationale|
|2016||5th CNAG Symposium on Genome Research: Single Cell Studies|
For publications that are currently unpublished or in-press, details are shown in italics.
Chapter (1 outputs)
|2016||Valdes-Mora F, Lee HJ, 'Single-Cell Genomics and Epigenomics', Essentials of Single-Cell Analysis Concepts, Applications and Future Prospects, Springer, Berlin Heidelberg (2016)|
Journal article (23 outputs)
Clark SJ, Smallwood SA, Lee HJ, Krueger F, Reik W, Kelsey G, 'Genome-wide base-resolution mapping of DNA methylation in single cells using single-cell bisulfite sequencing( scBS-seq)', NATURE PROTOCOLS, 12 534-U159 (2017)
Angermueller C, Lee HJ, Reik W, Stegle O, 'DeepCpG: Accurate prediction of single-cell DNA methylation states using deep learning', Genome Biology, 18 (2017)
Â© 2017 The Author(s). Recent technological advances have enabled DNA methylation to be assayed at single-cell resolution. However, current protocols are limited by incomplete CpG... [more]
Â© 2017 The Author(s). Recent technological advances have enabled DNA methylation to be assayed at single-cell resolution. However, current protocols are limited by incomplete CpG coverage and hence methods to predict missing methylation states are critical to enable genome-wide analyses. We report DeepCpG, a computational approach based on deep neural networks to predict methylation states in single cells. We evaluate DeepCpG on single-cell methylation data from five cell types generated using alternative sequencing protocols. DeepCpG yields substantially more accurate predictions than previous methods. Additionally, we show that the model parameters can be interpreted, thereby providing insights into how sequence composition affects methylation variabil ity.
Kalkan T, Olova N, Roode M, Mulas C, Lee HJ, Nett I, et al., 'Tracking the embryonic stem cell transition from ground state pluripotency', Development (Cambridge), 144 1221-1234 (2017)
Â© 2017. Published by The Company of Biologists Ltd. Mouse embryonic stem (ES) cells are locked into self-renewal by shielding from inductive cues. Release from this ground state ... [more]
Â© 2017. Published by The Company of Biologists Ltd. Mouse embryonic stem (ES) cells are locked into self-renewal by shielding from inductive cues. Release from this ground state in minimal conditions offers a system for delineating developmental progression from naÃ¯ve pluripotency. Here, we examine the initial transition process. The ES cell population behaves asynchronously. We therefore exploited a short-half-life Rex1::GFP reporter to isolate cells either side of exit from naÃ¯ve status. Extinction of ES cell identity in single cells is acute. It occurs only after near-complete elimination of naÃ¯ve pluripotency factors, but precedes appearance of lineage specification markers. Cells newly departed from the ES cell state display features of early post-implantation epiblast and are distinct from primed epiblast. They also exhibit a genome-wide increase in DNA methylation, intermediate between early and late epiblast. These findings are consistent with the proposition that naÃ¯ve cells transition to a distinct formative phase of pluripotency preparatory to lineage priming.
Gallego-Ortega D, Ledger A, Roden DL, Law AMK, Magenau A, Kikhtyak Z, et al., 'ELF5 Drives Lung Metastasis in Luminal Breast Cancer through Recruitment of Gr1+ CD11b+ Myeloid-Derived Suppressor Cells', PLoS Biology, 13 (2015)
Â© 2015 Gallego-Ortega et al. During pregnancy, the ETS transcription factor ELF5 establishes the milk-secreting alveolar cell lineage by driving a cell fate decision of the mamma... [more]
Â© 2015 Gallego-Ortega et al. During pregnancy, the ETS transcription factor ELF5 establishes the milk-secreting alveolar cell lineage by driving a cell fate decision of the mammary luminal progenitor cell. In breast cancer, ELF5 is a key transcriptional determinant of tumor subtype and has been implicated in the development of insensitivity to anti-estrogen therapy. In the mouse mammary tumor virus-Polyoma Middle T (MMTV-PyMT) model of luminal breast cancer, induction of ELF5 levels increased leukocyte infiltration, angiogenesis, and blood vessel permeability in primary tumors and greatly increased the size and number of lung metastasis. Myeloid-derived suppressor cells, a group of immature neutrophils recently identified as mediators of vasculogenesis and metastasis, were recruited to the tumor in response to ELF5. Depletion of these cells using specific Ly6G antibodies prevented ELF5 from driving vasculogenesis and metastasis. Expression signatures in luminal A breast cancers indicated that increased myeloid cell invasion and inflammation were correlated with ELF5 expression, and increased ELF5 immunohistochemical staining predicted much shorter metastasisÂ¿free and overall survival of luminal A patients, defining a group who experienced unexpectedly early disease progression. Thus, in the MMTV-PyMT mouse mammary model, increased ELF5 levels drive metastasis by co-opting the innate immune system. As ELF5 has been previously implicated in the development of antiestrogen resistance, this finding implicates ELF5 as a defining factor in the acquisition of the key aspects of the lethal phenotype in luminal A breast cancer.
Smallwood SA, Lee HJ, Angermueller C, Krueger F, Saadeh H, Peat J, et al., 'Single-cell genome-wide bisulfite sequencing for assessing epigenetic heterogeneity', Nature Methods, 11 817-820 (2014)
We report a single-cell bisulfite sequencing (scBSBS-seq) method that can be used to accurately measure DNADNADNA methylation at up to 48.4% of CpG sites. Embryonic stem cells gro... [more]
We report a single-cell bisulfite sequencing (scBSBS-seq) method that can be used to accurately measure DNADNADNA methylation at up to 48.4% of CpG sites. Embryonic stem cells grown in serum or in 2i medium displayed epigenetic heterogeneity, with '2i-like' cells present in serum culture. Integration of 12 individual mouse oocyte datasets largely recapitulated the whole DNADNADNA methylome, which makes scBSBS-seq a versatile tool to explore DNADNADNA methylation in rare cells and heterogeneous populations. Â© 2014 Nature America, Inc. All rights reserved.
Lee HJ, Hore TA, Reik W, 'Reprogramming the methylome: Erasing memory and creating diversity', Cell Stem Cell, 14 710-719 (2014)
The inheritance of epigenetic marks, in particular DNA methylation, provides a molecular memory that ensures faithful commitment to transcriptional programs during mammalian devel... [more]
The inheritance of epigenetic marks, in particular DNA methylation, provides a molecular memory that ensures faithful commitment to transcriptional programs during mammalian development. Epigenetic reprogramming results in global hypomethylation of the genome together with a profound loss of memory, which underlies naive pluripotency. Such global reprogramming occurs in primordial germ cells, early embryos, and embryonic stem cells where reciprocal molecular links connect the methylation machinery to pluripotency. Priming for differentiation is initiated upon exit from pluripotency, and we propose that epigenetic mechanisms create diversity of transcriptional states, which help with symmetry breaking during cell fate decisions and lineage commitment. Â© 2014 The Authors.
Lee HJ, Gallego-Ortega D, Ledger A, Schramek D, Joshi P, Szwarc MM, et al., 'Progesterone drives mammary secretory differentiation via RankL-mediated induction of Elf5 in luminal progenitor cells', Development (Cambridge), 140 1397-1401 (2013)
Progesterone-RankL paracrine signaling has been proposed as a driver of stem cell expansion in the mammary gland, and Elf5 is essential for the differentiation of mammary epitheli... [more]
Progesterone-RankL paracrine signaling has been proposed as a driver of stem cell expansion in the mammary gland, and Elf5 is essential for the differentiation of mammary epithelial progenitor cells. We demonstrate that Elf5 expression is induced by progesterone and that Elf5 and progesterone cooperate to promote alveolar development. The progesterone receptor and Elf5 are expressed in a mutually exclusive pattern, and we identify RankL as the paracrine mediator of the effects of progesterone on Elf5 expression in CD61+ progenitor cells and their consequent differentiation. Blockade of RankL action prevented progesterone-induced side branching and the expansion of Elf5 + mature luminal cells. These findings describe a mechanism by which steroid hormones can produce the expansion of steroid hormone receptor-negative mammary epithelial cells. Â© 2013. Published by The Company of Biologists Ltd.
Gallego-Ortega D, Oakes SR, Lee HJ, Piggin CL, Ormandy CJ, 'ELF5, normal mammary development and the heterogeneous phenotypes of breast cancer', Breast Cancer Management, 2 489-498 (2013)
Ficz G, Hore TA, Santos F, Lee HJ, Dean W, Arand J, et al., 'FGF signaling inhibition in ESCs drives rapid genome-wide demethylation to the epigenetic ground state of pluripotency', Cell Stem Cell, 13 351-359 (2013)
Genome-wide erasure of DNA methylation takes place in primordial germ cells (PGCs) and early embryos and is linked with pluripotency. Inhibition of Erk1/2 and Gsk3Ã signaling in ... [more]
Genome-wide erasure of DNA methylation takes place in primordial germ cells (PGCs) and early embryos and is linked with pluripotency. Inhibition of Erk1/2 and Gsk3Ã signaling in mouse embryonic stem cells (ESCs) by small-molecule inhibitors (called 2i) has recently been shown to induce hypomethylation. We show by whole-genome bisulphite sequencing that 2i induces rapid and genome-wide demethylation on a scale and pattern similar to that in migratory PGCs and early embryos. Major satellites, intracisternal A particles (IAPs), and imprinted genes remain relatively resistant to erasure. Demethylation involves oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), impaired maintenance of 5mC and 5hmC, and repression of the de novo methyltransferases (Dnmt3a and Dnmt3b) and Dnmt3L. We identify a Prdm14- and Nanog-binding cis-acting regulatory region in Dnmt3b that is highly responsive to signaling. These insights provide a framework for understanding how signaling pathways regulate reprogramming to an epigenetic ground state of pluripotency. Â© 2013 The Authors.
Lambert LJ, Walker S, Feltham J, Lee HJ, Reik W, Houseley J, 'Etoposide induces nuclear re-localisation of AID', PLoS ONE, 8 (2013)
During B cell activation, the DNA lesions that initiate somatic hypermutation and class switch recombination are introduced by activation-induced cytidine deaminase (AID). AID is ... [more]
During B cell activation, the DNA lesions that initiate somatic hypermutation and class switch recombination are introduced by activation-induced cytidine deaminase (AID). AID is a highly mutagenic protein that is maintained in the cytoplasm at steady state, however AID is shuttled across the nuclear membrane and the protein transiently present in the nucleus appears sufficient for targeted alteration of immunoglobulin loci. AID has been implicated in epigenetic reprogramming in primordial germ cells and cell fusions and in induced pluripotent stem cells (iPS cells), however AID expression in non-B cells is very low. We hypothesised that epige netic reprogramming would require a pathway that instigates prolonged nuclear residence of AID. Here we show that AID is completely re-localised to the nucleus during drug withdrawal following etoposide treatment, in the period in which double strand breaks (DSBs) are repaired. Re-localisation occurs 2-6 hours after etoposide treatment, and AID remains in the nucleus for 10 or more hours, during which time cells remain live and motile. Re-localisation is cell-cycle dependent and is only observed in G2. Analysis of DSB dynamics shows that AID is re-localised in response to etoposide treatment, however relocalisation occurs substantially after DSB formation and the levels of re-localisation do not correlate with Â¿H2AX levels. We conclude that DSB formation initiates a slow-acting pathway which allows stable long-term nuclear localisation of AID, and that such a pathway may enable AID-induced DNA demethylation during epigenetic reprogramming. Â© 2013 Lambert et al.
Lee HJ, Ormandy CJ, 'Interplay between progesterone and prolactin in mammary development and implications for breast cancer', Molecular and Cellular Endocrinology, 357 101-107 (2012)
Progesterone and prolactin remodel mammary morphology during pregnancy by acting on the mammary epithelial cell hierarchy. The roles of each hormone in mammary development have be... [more]
Progesterone and prolactin remodel mammary morphology during pregnancy by acting on the mammary epithelial cell hierarchy. The roles of each hormone in mammary development have been well studied, but evidence of signalling cross-talk between progesterone and prolactin is still emerging. Factors such as receptor activator of NFkB ligand (RANKL) may integrate signals from both hormones to orchestrate their joint actions on the epithelial cell hierarchy. Common targets of progesterone and prolactin signalling are also likely to integrate their pro-proliferative actions in breast cancer. Therefore, a thorough understanding of the interplay between progesterone and prolactin in mammary development may reveal therapeutic targets for breast cancer. This review summarises our understanding of Pg and PRL action in mammary gland development before focusing on molecular mechanisms of signalling cross-talk and the implications for breast cancer. Â© 2011 Elsevier Ireland Ltd.
Lee HJ, Ormandy CJ, 'Elf5, hormones and cell fate', Trends in Endocrinology and Metabolism, 23 292-298 (2012)
Recent elucidation of the stem and progenitor cell hierarchies that operate during normal tissue and organ development has provided a foundation for the development of new insight... [more]
Recent elucidation of the stem and progenitor cell hierarchies that operate during normal tissue and organ development has provided a foundation for the development of new insights into the disease process. These hierarchies are established by genetic mechanisms, which specify and determine cell fate and act as cell-clade gatekeepers, upon which all multicellular organisms depend for viability. Perturbation of this gatekeeper function characterizes developmentally based diseases, such as cancer. Here, the emerging gatekeeper and master regulatory roles of the ETS transcription factor Elf5 in several diverse developmental scenarios is reviewed, and how this function intersects with hormonal and growth factor mediated regulation of these processes is shown. Â© 2012 Elsevier Ltd.
Kalyuga M, Gallego-Ortega D, Lee HJ, Roden DL, Cowley MJ, Caldon CE, et al., 'ELF5 Suppresses Estrogen Sensitivity and Underpins the Acquisition of Antiestrogen Resistance in Luminal Breast Cancer', PLoS Biology, 10 (2012)
We have previously shown that during pregnancy the E-twenty-six (ETS) transcription factor ELF5 directs the differentiation of mammary progenitor cells toward the estrogen recepto... [more]
We have previously shown that during pregnancy the E-twenty-six (ETS) transcription factor ELF5 directs the differentiation of mammary progenitor cells toward the estrogen receptor (ER)-negative and milk producing cell lineage, raising the possibility that ELF5 may suppress the estrogen sensitivity of breast cancers. To test this we constructed inducible models of ELF5 expression in ER positive luminal breast cancer cells and interrogated them using transcript profiling and chromatin immunoprecipitation of DNA followed by DNA sequencing (ChIP-Seq). ELF5 suppressed ER and FOXA1 expression and broadly suppressed ER-driven patterns of gene expression including sets of genes distinguishing the luminal molecular subtype. Direct transcriptional targets of ELF5, which included FOXA1, EGFR, and MYC, accurately classified a large cohort of breast cancers into their intrinsic molecular subtypes, predicted ER status with high precision, and defined groups with differential prognosis. Knockdown of ELF5 in basal breast cancer cell lines suppressed basal patterns of gene expression and produced a shift in molecular subtype toward the claudin-low and normal-like groups. Luminal breast cancer cells that acquired resistance to the antiestrogen Tamoxifen showed greatly elevated levels of ELF5 and its transcriptional signature, and became dependent on ELF5 for proliferation, compared to the parental cells. Thus ELF5 provides a key transcriptional determinant of breast cancer molecular subtype by suppression of estrogen sensitivity in luminal breast cancer cells and promotion of basal characteristics in basal breast cancer cells, an action that may be utilised to acquire antiestrogen resistance. Â© 2012 Kalyuga et al.
Lee HJ, Hinshelwood RA, Bouras T, Gallego-Ortega D, ValdÃ©s-Mora F, Blazek K, et al., 'Lineage specific methylation of the Elf5 promoter in mammary epithelial cells', Stem Cells, 29 1611-1619 (2011)
Recent characterization of mammary stem and progenitor cells has improved our understanding of the transcriptional network that coordinates mammary development; however, little is... [more]
Recent characterization of mammary stem and progenitor cells has improved our understanding of the transcriptional network that coordinates mammary development; however, little is known about the mechanisms that enforce lineage commitment and prevent transdifferentiation in the mammary gland. The E-twenty six transcription factor Elf5 forces the differentiation of mammary luminal progenitor cells to establish the milk producing alveolar lineage. Methylation of the Elf5 promoter has been proposed to act as a lineage gatekeeper during embryonic development. We used bisulphite sequencing to investigate in detail whether Elf5 promoter methylation plays a role in lineage commitment during mammary development. An increase in Elf5 expression was associated with decreasing Elf5 promoter methylation in differentiating HC11 mammary cells. Similarly, purified mammary epithelial cells from mice had increased Elf5 expression and decreased promoter methylation during pregnancy. Finally, analysis of epithelial subpopulations revealed that the Elf5 promoter is methylated and silenced in the basal, stem cell-containing population relative to luminal cells. These results demonstrate that Elf5 promoter methylation is lineage-specific and developmentally regulated in the mammary gland in vivo, and suggest that loss of Elf5 methylation specifies the mammary luminal lineage, while continued Elf5 methylation maintains the stem cell and myoepithelial lineages. Â© AlphaMed Press.
Schramek D, Leibbrandt A, Sigl V, Kenner L, Pospisilik JA, Lee HJ, et al., 'Osteoclast differentiation factor RANKL controls development of progestin-driven mammary cancer', Nature, 468 98-102 (2010)
Breast cancer is one of the most common cancers in humans and will on average affect up to one in eight women in their lifetime in the United States and Europe. The Women's H... [more]
Breast cancer is one of the most common cancers in humans and will on average affect up to one in eight women in their lifetime in the United States and Europe. The Women's Health Initiative and the Million Women Study have shown that hormone replacement therapy is associated with an increased risk of incident and fatal breast cancer. In particular, synthetic progesterone derivatives (progestins) such as medroxyprogesterone acetate (MPA), used in millions of women for hormone replacement therapy and contraceptives, markedly increase the risk of developing breast cancer. Here we show that the in vivo administration of MPA triggers massive induction of the key osteoclast differentiation factor RANKL (receptor activator of NF-I Â°B ligand) in mammary-gland epithelial cells. Genetic inactivation of the RANKL receptor RANK in mammary-gland epithelial cells prevents MPA-induced epithelial proliferation, impairs expansion of the CD49f hi stem-cell-enriched population, and sensitizes these cells to DNA-damage-induced cell death. Deletion of RANK from the mammary epithelium results in a markedly decreased incidence and delayed onset of MPA-driven mammary cancer. These data show that the RANKL/RANK system controls the incidence and onset of progestin-driven breast cancer. Â© 2010 Macmillan Publishers Limited. All rights reserved.
Hilton HN, Kalyuga M, Cowley MJ, Alles MC, Lee HJ, Caldon CE, et al., 'The antiproliferative effects of progestins in T47D breast cancer cells are tempered by progestin induction of the ETS transcription factor Elf5', Molecular Endocrinology, 24 1380-1392 (2010)
Prolactin and progesterone act together to regulate mammary alveolar development, and both hormones have been implicated in breast cancer initiation and progression. Here we show ... [more]
Prolactin and progesterone act together to regulate mammary alveolar development, and both hormones have been implicated in breast cancer initiation and progression. Here we show that Elf5, a prolactin-induced ETS transcription factor that specifies the mammary secretory cell lineage, is also induced by progestins in breast cancer cells via a direct mechanism. To define the transcriptional response to progestin elicited via Elf5, we made an inducible Elf5 short hairpin-RNA knock-down model in T47D breast cancer cells and used it to prevent the progestin-induction of Elf5. Functional analysis of Affymetrix gene expression data using Gene Ontologies and Gene Set Enrichment Analysis showed enhancement of the progestin effects on cell cycle gene expression. Cell proliferation assays showed a more efficacious progestin-induced growth arrest when Elf5 was kept at baseline levels. These results showed that progestin induction of Elf5 expression tempered the antiproliferative effects of progestins in T47D cells, providing a further mechanistic link between prolactin and progestin in the regulation of mammary cell phenotype. Copyright Â© 2010 by The Endocrine Society.
Menzies KK, Lee HJ, LefÃ¨vre C, Ormandy CJ, Macmillan KL, Nicholas KR, 'Insulin, a key regulator of hormone responsive milk protein synthesis during lactogenesis in murine mammary explants', Functional and Integrative Genomics, 10 87-95 (2010)
Murine milk protein gene expression requires insulin, hydrocortisone, and prolactin; however, the role of insulin is not well understood. This study, therefore, examined the requi... [more]
Murine milk protein gene expression requires insulin, hydrocortisone, and prolactin; however, the role of insulin is not well understood. This study, therefore, examined the requirement of insulin for milk protein synthesis. Mammary explants were cultured in various combinations of the lactogenic hormones and global changes in gene expression analysed using Affymetrix microarray. The expression of 164 genes was responsive to insulin, and 18 were involved in protein synthesis at the level of transcription and posttranscription, as well as amino acid uptake and metabolism. The folate receptor gene was increased by fivefold, highlighting a potentially important role for the hormone in folate metabolism, a process that is emerging to be central for protein synthesis. Interestingly, gene expression of two milk protein transcription factors, Stat5a and Elf5, previously identified as key components of prolactin signalling, both showed an essential requirement for insulin. Subsequent experiments in HCll cells confirmed that Stat5a and Elf5 gene expression could be induced in the absence of prolactin but in the presence of insulin. Whereas prolactin plays an essential role in phosphorylating and activating Stat5a, gene expression is only induced when insulin is present. This indicates insulin plays a crucial role in the transcription of the milk protein genes. Â© 2010 Springer-Verlag.
Leung KC, Brce J, Doyle N, Lee HJ, Leong GM, SjÃ¶gren K, Ho KKY, 'Regulation of growth hormone signaling by selective estrogen receptor modulators occurs through suppression of protein tyrosine phosphatases', Endocrinology, 148 2417-2423 (2007)
Activation of the Janus kinase 2 (JAK2)/signal transducer and activator of transcription 5 (STAT5) pathway by GH is terminated by the suppressors of cytokine signaling (SOCSs) and... [more]
Activation of the Janus kinase 2 (JAK2)/signal transducer and activator of transcription 5 (STAT5) pathway by GH is terminated by the suppressors of cytokine signaling (SOCSs) and protein tyrosine phosphatases, Src homology 2 domain-containing protein tyrosine phosphatase (SHP)-1 and SHP-2. Based on our recent report that estrogen inhibits GH signaling by stimulating SOCS-2 expression, we investigated the effects of selective estrogen receptor modulators (SERMs) on GH signaling in human embryonic kidney (HEK293) and breast cancer (MDA-MB-231) cells expressing human GH receptor and estrogen receptor-a. 17Ã-Estradiol (E 2 ) suppressed GH activation of a STAT5-responsive luciferase reporter and JAK2 phosphorylation in both cell models. 4-Hydroxytamoxifen and raloxifene augmented these actions of GH in HEK293 cells but not breast cancer cells. SOCS-2 expression in both cell types was stimulated by E 2 but unaffected by SERMs. In HEK293 cells, SHP-1 was inhibited by raloxifene and 4-hydroxytamoxifen, whereas the latter additionally inhibited SHP-2. The phosphatases were unaffected by E 2 . In breast cancer cells, phosphatase activity was not altered by SERMs or E 2 . In summary, estrogen inhibited the JAK2/STAT5 signaling of GH and stimulated SOCS-2 expression in both HEK293 and breast cancer cells. By contrast, SERMs augmented GH signaling by reducing SHP activities in HEK293 cells and had no effect on both in breast cancer cells. We provide the first evidence for a novel mechanism regulating GH signaling, in which SERMs enhance GH activation of the JAK2/STAT5 pathway in a cell-type-dependent manner by attenuating protein tyrosine phosphatase activities. Copyright Â© 2007 by The Endocrine Society.
Lee HJ, Mun HC, Lewis NC, Crouch MF, Culverston EL, Mason RS, Conigrave AD, 'Allosteric activation of the extracellular Ca2+-sensing receptor by L-amino acids enhances ERK1/2 phosphorylation', Biochemical Journal, 404 141-149 (2007)
The calcium-sensing receptor (CaR) mediates feedback control of Ca 2+ o (extracellular Ca 2+ ) concentration. Although the mechanisms are not fully understood, the CaR couples t... [more]
The calcium-sensing receptor (CaR) mediates feedback control of Ca 2+ o (extracellular Ca 2+ ) concentration. Although the mechanisms are not fully understood, the CaR couples to several important intracellular signalling enzymes, including PI-PLC (phosphoinositide-specific phospholipase C), leading to Ca 2+ i (intracellular Ca 2+ ) mobilization, and ERK1/2 (extracellular-signal-regulated kinase 1/2). In addition to Ca 2+ o , the CaR is activated allosterically by several subclasses of L-amino acids, including the aromatics L-phenylalanine and L-tryptophan. These amino acids enhance the Ca 2+ o -sensitivity of Ca 2+ i mobilization in CaR-expressing HEK-293 (human embryonic kidney) cells and normal human parathyroid cells. Furthermore, on a background of a physiological fasting serum L-amino acid mixture, they induce a small, but physiologically significant, enhancement of Ca 2+ o -dependent suppression of PTH (parathyroid hormone) secretion. The impact of amino acids on CaR-stimulated ERK1/2, however, has not been determined. In the present study, we examined the effects of L-amino acids on Ca 2+ o -stimulated ERK1/2 phosphorylation as determined by Western blotting and a newly developed quantitative assay (SureFire). L-Amino acids induced a small, but significant, enhancement of Ca 2+ o -stimulated ERK1/2. In CaR-expressing HEK-293 cells, 10 mM L-phenylalanine lowered the EC 50 for Ca 2+ o from approx. 2.3 to 2.0 mM in the Western blot assay and from 3.4 to 2.9 mM in the SureFire assay. The effect was stereoselective (L > D), and another aromatic amino acid, L-tryptophan, was also effective. The effects of amino acids were investigated further in HEK-293 cells that expressed the CaR mutant S169T. L-Phenylalanine normalized the EC 50 for Ca 2+ o -stimulated Ca 2+ i mobilization from approx. 12 mM to 5.0 mM and ERK1/2 phosphorylation from approx. 4.6 mM to 2.6 mM. Taken together, the data indicate that L-phenylalanine and other amino acids enhance the Ca 2+ o -sensitivity of CaR-stimulated ERK1/2 phosphorylation; however, the effect is comparatively small and operates in the form of a fine-tuning mechanism. Â© The Authors.
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Dr Heather Lee
Information Based Medicine
School of Biomedical Sciences and Pharmacy
Faculty of Health and Medicine