Geoffrey Lewis Hammond

Professor

Relevant Degree Programs

 

Graduate Student Supervision

Doctoral Student Supervision (Jan 2008 - May 2019)
Corticosteroid-Binding Globulin (CBG) : deficiencies and the role of CBG in disease processes (2017)

Corticosteroid-binding globulin (CBG, SERPINA6) is a serine protease inhibitor family member produced by hepatocytes. Plasma CBG transports biologically active glucocorticoids, determines their bioavailability to target tissues and acts as an acute-phase negative protein with a role in the delivery of glucocorticoids to sites of inflammation. A few CBG-deficient individuals have been identified, yet the clinical significance of this remain unclear. In this thesis, I investigated 1) the biochemical consequences of naturally occurring single nucleotide polymorphisms in the SERPINA6 gene, 2) the role of human CBG during infections and acute inflammation and 3) CBG as a biomarker of inflammation in rats. A comprehensive analysis of functionally relevant naturally occurring SERPINA6 SNP revealed 11 CBG variants with abnormal production and/or function, diminished responses to proteolytic cleavage of the CBG reactive center loop (RCL) or altered recognition by monoclonal antibodies. In a genome-wide association study, plasma cortisol levels were most closely associated with SERPINA6 SNPs and plasma CBG-cortisol binding capacity. These studies indicate that human CBG variants need to be considered in clinical evaluations of patients with abnormal cortisol levels. In addition, I obtained evidence that discrepancies in CBG values obtained by the 9G12 ELISA compared to CBG binding capacity and 12G2 ELISA are likely due to differential N-glycosylation rather than proteolysis, as recently reported. In relation to human inflammation, the bacterial protease Pseudomonas aeruginosa elastase was shown to cleave the RCL and α-2-macroglobulin specifically inhibited this. ICU patients with a variety of illnesses had significantly reduced plasma CBG levels, with the lowest levels in individuals with severe inflammation. Similar results were observed in a rodent model of inflammation, where significant reductions in plasma CBG levels were associated with CBG proteolysis and the down-regulation of hepatic Serpina6 expression. In addition, lower baseline plasma CBG levels in Harlan Sprague Dawley rats were linked with an increased susceptibility to inflammation. Together, the human and rodent studies highlight the importance of CBG in inflammatory reactions and suggest that CBG is a useful biomarker of inflammation onset and severity.

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Functional characterization of sex hormone-binding globulin genetic polymorphism (2015)

Human plasma SHBG is produced by the liver, and it transports biologically active sex steroids and determines their availability to target tissues. The 4.3 kb human SHBG transcriptional unit encoding a signal polypeptide for secretion followed by two laminin G (LG)-like domains is utilized by hepatocytes. The N-terminal LG domain of SHBG contains a region responsible for homodimer formation, and a steroid ligand-binding site that accommodates androgens and estrogens in opposite orientations. Among over 250 genetic polymorphisms identified in human SHBG, few are functionally characterized. In this research, I have performed a comprehensive analysis of functionally relevant SHBG single nucleotide polymorphisms (SNPs). Nine out of nineteen non-synonymous SNPs within the coding region of SHBG N-terminal LG domain were shown to encode SHBG mutants with abnormal properties in steroid ligand binding, calcium coordination, fibulin-2 interaction, glycosylation or secretion. In particular, SHBG R123H (encoded by rs143269613) has a general reduction in affinity for steroid ligands, whereas SHBG E176K (encoded by rs372114420) has a higher affinity specifically for estradiol. Crystallography revealed that instead of losing the structural integrity of the steroid-binding site, reduced flexibility of the loop region that covers the steroid-binding site, and conformational changes at the opening rim of a putative estradiol entrance, likely account for the abnormal steroid-binding affinities of SHBG R123H and SHBG E176K, respectively. Among eight SNPs within SHBG regulatory sequences selected for analysis, only rs138097069 increases SHBG promoter activity. In silico prediction revealed that rs138097069 is located within a putative FXR binding site, while rs6257, which is linked to low plasma SHBG concentrations, is located within a putative FOXA2 binding element. In HepG2 cells, GW4064-activated FXR and overexpressed FOXA2 both suppress SHBG expression by direct binding to their corresponding binding elements in an HNF4⍺-independent manner. By contrast, knock-down of FXR reduces, while knock-down of FOXA2 induces, HNF4⍺ expression and SHBG production. Characterization of functional SHBG SNPs has provided molecular explanations of how genetic differences contribute to SHBG production and function, and has identified possible roles for two novel regulators, FXR and FOXA2, in a more complex regulatory network that determines SHBG expression.

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Regulated expression of aggrecanases of ADAMTS family in endometrial physiology and pathology (2011)

The ADAMTS (A Disintegrin and Metalloproteinase with TromboSpondin Repeats) are a novel family of secreted metalloproteinases. There is increasing evidence that distinct ADAMTS subtypes play key roles in embryonic development, reproduction and cancer. Nineteen ADAMTS subtypes have been identified in humans but most of them have been characterized only at the structural level. ADAMTS-1, -4, -5, -8, -9 and -15 have been subclassified into a subfamily, known as aggrecanases, owing to their ability to cleave the important extracellular matrix components, versican and aggrecan. Previous studies have determined that ADAMTS-1 and -5 are expressed in first trimester decidual cells and are regulated by IL-1ß and TGF- ß1. I have now found that gonadal steroids have complex regulatory effects upon ADAMTS-1 mRNA and ADAMTS-1 levels in endometrial stromal cells during the human menstrual cycle. I further demonstrate that progesterone (P4) and 5α-dihydrotestosterone (DHT), differentially regulated ADAMTS-5, -8, and -9 mRNA and protein levels in human endometrial stromal cells, suggesting that aggrecanases contribute to steroid-mediated ECM remodeling in the endometrium in preparation for pregnancy. My loss- and gain- of function studies have confirmed a function for ADAMTS-1 in endometrial cancer invasion. Overexpression of ADAMTS-1 in well-differentiated ECC-1 endometrial carcinoma cells promoted cell invasion. In contrast, siRNA-mediated silencing of endogenous ADAMTS-1 in poorly differentiated KLE cells decreased their invasive capacity. I have also found that 17ß-estradiol (E2) can up-regulate ADAMTS-1 mRNA and protein levels in ECC-1 cells. This suggests that ADAMTS-1 plays an important role in endometrial cancer progression, and that E2 promotes well-differentiated endometrial cancer cell invasion, at least in part by specific up-regulating ADAMTS-1 expression. Overall, my research provides useful insight into the molecular mechanisms that regulate endometrial physiology and pathology, and additional support for the concept that ADAMTS represent potentially useful prognostic biomarkers of recurrent pregnancy loss and endometrial cancer.

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Regulation of androgen action by sex hormone-binding globulin (2011)

Sex hormone-binding globulin (SHBG) binds androgens and estrogens with high affinity, and regulates the distribution of these sex steroids in the blood and other biological fluids. Liver is the primary site of SHBG production, but the human SHBG transcription unit responsible for this is also expressed in proximal convoluted tubule (PCT) epithelial cells in a transgenic mouse model. Unlike hepatocytes which actively secrete SHBG and retain little immunoreactive SHBG within their cytoplasm, an incompletely glycosylated SHBG isoform has been found to accumulate inside PCT cells. These cells are androgen target cells, and the presence of SHBG within them accentuates androgen-dependent regulation of gene expression mediated by the androgen receptor (AR). The retention and accumulation of SHBG within cells involves an interaction with the intracellular 37-kDa laminin receptor precursor (LRP), which was identified as a SHBG interacting-protein using yeast hybrid screen. This interaction was confirmed by Glutathione S-transferase pull-down assays, and was prevented by mutating the LRP laminin-binding site. Human SHBG was found to co-localize LRP in the peri-nuclear location of renal epithelial cells of transgenic mice and SHBG:LRP complexes were observed within the endoplasmic reticulum of these cells by immunoprecipitation assay. These data suggest that a physical interaction between SHBG and LRP contributes to the intracellular trapping of an incompletely glycosylated SHBG in PCT cells and an increase in AR-mediated androgen action. Since human SHBG transcripts have been reported in prostate cancer cells, we examined whether SHBG in the extracellular environment or the cytoplasm of human LNCaP prostate cancer cells influences their response to androgens. Although human SHBG is cleaved into two laminin G-like (LG) domains by kallikrein-related peptidase 4, this does not change its steroid-binding activity. Furthermore, the amino-terminal SHBG LG domiain induced AR-mediated androgen activity in LNCaP cells in the same way as intact SHBG. Although immunoreactive SHBG was not detected within LNCaP cells, siRNA-mediated knockdown of SHBG transcripts decreased AR-dependent inceases in prostate-specific antigen mRNA levels and androgen-reporter gene activity. These data suggest that while SHBG in LNCaP cell medium limits the metabolic clearance of androgens, SHBG within these cells enhances AR-induced gene expression.

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The characterization of ADAMTS-12 in the regulation of human trophoblast invasion in vitro (2011)

The distintegrin-like and metalloproteinase with thrombospondin repeats (ADAMTS) are members of a gene family of secreted, multidomain and multifunctional proteinases that are able to proteolytically degrade a diverse array of cellular, extracellular and extracellular matrix (ECM) substrates. We examined the presence of ADAMTS in first trimester human placenta and only ADAMTS-12 was present in cultures of invasive extravillous cytotrophoblast (EVT) at significantly higher levels than in poorly invasive JEG-3 chorocarcinoma cells. Immunohistochemistry staining of chorionic villi derived from first trimester human placenta demonstrated that ADAMTS-12 was intensively immunolocalized in the cytotrophoblast layer but weakly immunolocalized in the poorly-invasive syncytial trophoblast layer. Gonadotropin-releasing hormone (GnRH)-I and -II increased ADAMTS-12 expression in EVT in time- and concentration-dependent manners, and these two hormones exert functions through different pathways. Loss- or gain-of function studies using siRNA and stable transfection strategies demonstrated that the ADAMTS-12 promotes trophoblast invasion. Surprisingly, this function of ADAMTS-12 is independent of its catalytic activity. C-terminal sequential deletions of ADAMTS-12 demonstrated that the disintegrin-like domain plays crucial role in cellular localization of ADAMTS-12. Laminin-5 is a component of the ECM, influencing cell migration and adhesion. The disintegrin-like domain and ancillary domains of ADAMTS-12 are associated with increased laminin-5 expression in JEG-3 cells through the activation of the ERK/MAPK signaling pathway. The integrin expression repertoire is also modified by ADAMTS-12. In particular, laminin-5 receptor integrin α6β4 is increased by the exogenous expression of ADAMTS-12 in JEG-3 cells.Together, these data support a novel hypothesis that ADAMTS-12 plays a non-redundant role in human trophoblastic cell invasion, which is independent of its catalytic activity but dependent on the disintegrin-like domain and ancillary domains. This role involves alteration of cell-ECM interactions, which leads to a reduction of cell adhesion capability and promotes cell invasion.

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Cadherin-linked molecular mechanisms governing the terminal differentiation of human trophoblastic cells in vitro (2010)

Background: The formation of the multinucleated syncytial trophoblast of the human placenta is a critical step in pregnancy, which is prone to failure. In these studies, I have examined the role of TWIST, a transcription factor identified as a key repressor of E-cad expression in normal and cancer cells of diverse origins, in the differentiation of human trophoblastic cells in vitro. The invasion of extravillous cytotrophoblasts (EVTs) into the underlying maternal tissues and vasculature is a key step in human placentation. The molecular mechanisms underlying the development of the invasive phenotype of EVTs include many of those first identified as having a role in cancer cell metastasis. In view of these observations, I have examined the expression, regulation, and function of Twist, Runx2 and N-cad in human trophoblastic cells in vitro. Materials and Methods: Gain or loss-of-function studies were then performed to determine the role of Twist in terminal differentiation and fusion in these cells. The presence of multinucleated syncytium was confirmed by indirect immunofluorescence. Concentration- and time-dependent studies were performed to determine whether interleukin (IL)-1β and transforming growth factor (TGF)-β1 regulate Twist and Runx2 mRNA and protein levels in EVTs. Next, a siRNA strategy was employed to determine the role of Twist, Runx2 and N-cad in HTR-8/SVneo EVT cells. Results: Exogenous expression of Twist resulted in a continuous and progressive decrease in E-cad expression and the subsequent formation of syncytium in BeWo cells maintained under normal culture conditions. In contrast, siRNA specific for Twist inhibited the cAMP-mediated differentiation of these cells over time in culture. The cytokines, IL-1β and TGF-β1, respectively induced the differential up- and down-regulation of Twist and Runx2 expression in primary cultures of EVTs in both a concentration and time-dependent manner. Use of a siRNA strategy demonstrated that a reduction in Twist, Runx2 or N-cad in HTR-8/SVneo cells concomitantly decreased the invasiveness of these cells. Conclusions: Collectively, my findings demonstrate that TWIST is an upstream regulator of the E-CAD-mediated terminal differentiation and fusion of human trophoblastic cells in vitro. TWIST, RUNX2 and N-CAD are key molecules underlying the invasive capacity of EVTs.

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