Eliza Whiteside

Matrix metalloproteinase-9 and tissue inhibitor of metalloproteinase-3 are key regulators of extracellular matrix degradation by mouse embryos

Abstract Embryo implantation in humans and rodents is a highly invasive yet tightly controlled process involving extracellular matrix (ECM) degradation. Matrix metalloproteinase 9 (MMP-9) has been implicated as the major facilitator of this ECM degradation. MMP-9 is expressed by the embryos trophoblast cells, whereas tissue inhibitor of metalloproteinases 3 (TIMP-3) is expressed by the maternal uterine cells immediately adjacent to the trophoblast. We examined the functional roles of MMP-9 and TIMP-3 during in vitro ECM degradation by mouse embryos. Blastocysts were treated with either MMP-9 antisense or sense oligonucleotides and incubated on an ECM gel. The extent of ECM degradation exhibited by the blastocysts due to proteinase secretion was quantified. Embryos exposed to MMP-9 antisense oligonucleotides exhibited reduced ECM-degrading activity as compared with controls, and this reduced activity was correlated with the level of MMP-9 secreted by the embryos. The functional role of TIMP-3 was then examined by incubating blastocysts on an ECM gel that had been impregnated with various amounts of TIMP-3. In a dose-dependent manner, increases in TIMP-3 resulted in a reduction in ECM degradation and were correlated with diminished MMP-9 activity. These results provide important functional evidence that in vitro ECM degradation is regulated by embryo-derived MMP-9 and ECM-derived TIMP-3.

Ghrelin and cancer.

Ghrelin is a peptide hormone that was originally isolated from the stomach as the endogenous ligand for the growth hormone secretagogue receptor (GHSR). Ghrelin has many functions, including the regulation of appetite and gut motility, growth hormone release from the anterior pituitary and roles in the cardiovascular and immune systems. Ghrelin and its receptor are expressed in a number of cancers and cancer cell lines and may play a role in processes associated with cancer progression, including cell proliferation, apoptosis, and cell invasion and migration.

Cloning of a novel insulin-regulated ghrelin transcript in prostate cancer.

Ghrelin is a multifunctional hormone, with roles in stimulating appetite and regulating energy balance, insulin secretion and glucose homoeostasis. The ghrelin gene locus (GHRL) is highly complex and gives rise to a range of novel transcripts derived from alternative first exons and internally spliced exons. The wild-type transcript encodes a 117 amino acid preprohormone that is processed to yield the 28 amino acid peptide ghrelin. Here, we identified insulin-responsive transcription corresponding to cryptic exons in intron 2 of the human ghrelin gene. A transcript, termed in2c-ghrelin (intron 2-cryptic), was cloned from the testis and the LNCaP prostate cancer cell line. This transcript may encode an 83 amino acid preproghrelin isoform that codes for ghrelin, but not obestatin. It is expressed in a limited number of normal tissues and in tumours of the prostate, testis, breast and ovary. Finally, we confirmed that in2c-ghrelin transcript expression, as well as the recently described in1-ghrelin transcript, is significantly upregulated by insulin in cultured prostate cancer cells. Metabolic syndrome and hyperinsulinaemia have been associated with prostate cancer risk and progression. This may be particularly significant after androgen deprivation therapy for prostate cancer, which induces hyperinsulinaemia, and this could contribute to castrate-resistant prostate cancer growth. We have previously demonstrated that ghrelin stimulates prostate cancer cell line proliferation in vitro. This study is the first description of insulin regulation of a ghrelin transcript in cancer and should provide further impetus for studies into the expression, regulation and function of ghrelin gene products.

A longitudinal study of the impact of chronic psychological stress on health-related quality of life and clinical biomarkers: protocol for the Australian Healthy Aging of Women Study

BackgroundDespite advancements in our understanding of the importance of stress reduction in achieving good health, we still only have limited insight into the impact of stress on cellular function. Recent studies have suggested that exposure to prolonged psychological stress may alter an individual’s physiological responses, and contribute to morbidity and mortality. This paper presents an overview of the study protocol we are using to examine the impact of life stressors on lifestyle factors, health-related quality of life and novel and established biomarkers of stress in midlife and older Australian women.The primary aim of this study is to explore the links between chronic psychological stress on both subjective and objective health markers in midlife and older Australian women. The study examines the extent to which exposure frightening, upsetting or stressful events such as natural disasters, illness or death of a relative, miscarriage and relationship conflict is correlated with a variety of objective and subjective health markers.Methods/DesignThis study is embedded within the longitudinal Healthy Aging of Women’s study which has collected data from midlife and older Australian women at 5 yearly intervals since 2001, and uses the Allostastic model of women’s health by Groër and colleagues in 2010. The current study expands the focus of the HOW study and will assess the impact of life stressors on quality of life and clinical biomarkers in midlife and older Australian women to explain the impact of chronic psychological stress in women.DiscussionThe proposed study hypothesizes that women are at increased risk of exposure to multiple or repeated stressors, some being unique to women, and the frequency and chronicity of stressors increases women’s risk of adverse health outcomes. This study aims to further our understanding of the relationships between stressful life experiences, perceived quality of life, stress biomarkers, chronic illness, and health status in women.

Urokinase-type plasminogen activator (uPA) and matrix metalloproteinase-9 (MMP-9) expression and activity during early embryo development in the cow

Several extracellular matrix (ECM)-degrading proteinases are hypothesised to play important roles during early mammalian development. In particular, urokinase-type plasminogen activator (uPA) and matrix metalloproteinase 9 (MMP-9) are expressed in peri-implantation mouse, sheep, and pig embryos and are implicated in the implantation process. These proteinases are not expressed in early (pre-blastocyst) mouse, sheep or pig embryos. The aim of this study was to establish the gene expression and proteolytic activity of uPA and MMP-9 in in vitro-produced (IVP) cow embryos. Using RT-PCR, mRNA transcripts for uPA and MMP-9 were detected during the first 7 days of development. To investigate the activity of these proteinases, conditioned media from various stages of development (days 2, 3, 4, 5 and 7) were assayed for uPA activity by chromogenic assay and MMP-9 activity by gelatin zymography. Both uPA and MMP-9 activities were detected in the media samples indicating the production and secretion of these proteinases. This pattern of proteinase expression is novel in comparison to the mouse where uPA and MMP-9 are only expressed from the blastocyst stage onwards. The results of this study suggest that these ECM proteinases have a role prior to implantation in the cow, in contrast to that exhibited by mouse, sheep and pig embryos.

Multi-species sequence comparison reveals conservation of ghrelin gene-derived splice variants encoding a truncated ghrelin peptide

The peptide hormone ghrelin is a potent orexigen produced predominantly in the stomach. It has a number of other biological actions, including roles in appetite stimulation, energy balance, the stimulation of growth hormone release and the regulation of cell proliferation. Recently, several ghrelin gene splice variants have been described. Here, we attempted to identify conserved alternative splicing of the ghrelin gene by cross-species sequence comparisons. We identified a novel human exon 2-deleted variant and provide preliminary evidence that this splice variant and in1-ghrelin encode a C-terminally truncated form of the ghrelin peptide, termed minighrelin. These variants are expressed in humans and mice, demonstrating conservation of alternative splicing spanning 90 million years. Minighrelin appears to have similar actions to full-length ghrelin, as treatment with exogenous minighrelin peptide stimulates appetite and feeding in mice. Forced expression of the exon 2-deleted preproghrelin variant mirrors the effect of the canonical preproghrelin, stimulating cell proliferation and migration in the PC3 prostate cancer cell line. This is the first study to characterise an exon 2-deleted preproghrelin variant and to demonstrate sequence conservation of ghrelin gene-derived splice variants that encode a truncated ghrelin peptide. This adds further impetus for studies into the alternative splicing of the ghrelin gene and the function of novel ghrelin peptides in vertebrates.

The gut-brain axis, the human gut microbiota and their integration in the development of obesity

Obesity is a global epidemic, placing socioeconomic strain on public healthcare systems, especially within the so-called Western countries, such as Australia, United States, United Kingdom, and Canada. Obesity results from an imbalance between energy intake and energy expenditure, where energy intake exceeds expenditure. Current non-invasive treatments lack efficacy in combating obesity, suggesting that obesity is a multi-faceted and more complex disease than previously thought. This has led to an increase in research exploring energy homeostasis and the discovery of a complex bidirectional communication axis referred to as the gut-brain axis. The gut-brain axis is comprised of various neurohumoral components that allow the gut and brain to communicate with each other. Communication occurs within the axis via local, paracrine and/or endocrine mechanisms involving a variety of gut-derived peptides produced from enteroendocrine cells (EECs), including glucagon-like peptide 1 (GLP1), cholecystokinin (CCK), peptide YY3−36 (PYY), pancreatic polypeptide (PP), and oxyntomodulin. Neural networks, such as the enteric nervous system (ENS) and vagus nerve also convey information within the gut-brain axis. Emerging evidence suggests the human gut microbiota, a complex ecosystem residing in the gastrointestinal tract (GIT), may influence weight-gain through several inter-dependent pathways including energy harvesting, short-chain fatty-acids (SCFA) signalling, behaviour modifications, controlling satiety and modulating inflammatory responses within the host. Hence, the gut-brain axis, the microbiota and the link between these elements and the role each plays in either promoting or regulating energy and thereby contributing to obesity will be explored in this review.

Location of Residence and Breast Cancer Stage: A SEER Population‐based Analysis

To the Editor: Location of residence may influence breast cancer risk, with several reports indicating that living in locations closer to the equator may reduce both incidence and mortality from breast cancer (1–4). For example, a recent, large French cohort study found that women who resided at southern latitudes (<46° N) had a 10% decreased risk of breast cancer compared to women living at northern latitudes (>49° N) (1). A combined analysis involving 175 countries found a dose-response relationship between breast cancer incidence rates and latitude, with rates increasing as the distance from the equator increased (R = 0.43; p < 0.001) (2). Furthermore, women over 50 years old living in the Northeast of the U.S. were found to have a 13% increased risk of breast cancer mortality compared to women in the South (4). While these studies have examined the association between location of residence and breast cancer incidence and mortality, to our knowledge no research has examined the association between location of residence and breast cancer stage at diagnosis. We investigated whether there was an association between location of residence and breast cancer stage at diagnosis in the U.S., hypothesizing that women living in northern latitudes of the U.S. (>37° N) would have an increased risk of being diagnosed with advanced breast cancer compared to women living in southern latitudes (≤37° N). We used the Surveillance, Epidemiology, and End Results (SEER) data, a population-based database containing 18 cancer registries that cover 28% of the U.S. population, to identify invasive cases of breast cancer (n = 490,463) in women aged 20 years and older diagnosed from 2000 to 2010. Stage was dichotomized into “localized” and “advanced,” where localized stage refers to cancers that have not spread past the site of origin (breast) and advanced stage is defined as cancer spread beyond the local site (regional and distant breast cancer metastases). Location of residence at the time of diagnosis was defined by latitude and dichotomized into “North” (>37° N) and “South” (≤37° N). County-level variables were used for population demographics and in the analyses as potential confounders and included educational attainment, families below poverty, unemployment, foreign born, household income, mammography, and rurality. County-level variables are not individually based; rather, classification is assigned to individuals and reflects the probability that an individual from a specific county would have such characteristics. In the adjusted analysis we found the risk of being diagnosed with advanced breast cancer was significantly associated with younger age (OR = 2.49; 95% CI: 2.29, 2.70), ER-negative (OR = 1.12; 95% CI: 1.10, 1.14) and PR-negative (OR = 1.19; 95% CI: 1.17, 1.21) breast cancers, and being black (OR = 1.32; 95% CI: 1.30, 1.35). While in the crude analysis we found women living in the north were about 8% less likely (OR = 0.92; 95% CI: 0.91, 0.93) to be diagnosed with advanced breast cancer, this association did not remain significant in the adjusted model. However, when we stratified by race, we found that, in addition to an increased risk of being diagnosed with advanced breast cancer for younger age and PR-negative status, black women living in the North (>37° N) were 7% (OR = 0.93; 95% CI: 0.88, 0.98) less likely to be diagnosed with advanced breast cancer compared to black women living in the South (≤37° N) (Table 1). To our knowledge, this is the first study of this scale to examine the relationship of location of residence and breast cancer stage at diagnosis. Previous studies have investigated location of residence and breast cancer risk (1–3,5–7); however, the results of such studies have been inconsistent. Some indicated no association between location of residence and risk of breast cancer (5–7), while others found that individuals living in Address correspondence and reprint requests to Lindsay Brandon, MPH, Institute of Health and Biomedical Innovation, Queensland University of Technology, 60 Musk Ave, Kelvin Grove, QLD 4059, Australia, or e-mail: lindsay.brandon@qut.edu.au