Andrew B. Bicknell

Characterization of a Serine Protease that Cleaves Pro-γ-Melanotropin at the Adrenal to Stimulate Growth

The adrenal gland requires stimuli from peptides derived from the ACTH precursor, pro-opiomelanocortin (POMC), to maintain its tonic state. Studies have proposed that a specific postsecretional cleavage of the nonmitogenic N-terminal 16 kDa fragment, also known as pro-gamma-melanotropin (pro-gamma-MSH), is required, releasing shorter fragments that promote adrenal growth. Here, we provide evidence for this hypothesis by the cloning and characterization of a serine protease that is upregulated during growth of the adrenal cortex. It is expressed exclusively in the outer adrenal cortex, the site of cell proliferation, and in the Y1 adrenal cell line. We also show that it is required for growth of Y1 cells, remains bound to the cell surface, and cleaves its substrate, pro-gamma-MSH, at a specific bond.

The tissue-specific processing of pro-opiomelanocortin.

It is just over 30 years since the definitive identification of the adrenocorticotrophin (ACTH) precursor, pro‐opiomelanocotin (POMC). Although first characterised in the anterior and intermediate lobes of the pituitary, POMC is also expressed in a number of both central and peripheral tissues including the skin, central nervous tissue and placenta. Following synthesis, POMC undergoes extensive post‐translational processing producing not only ACTH, but also a number of other biologically active peptides. The extent and pattern of this processing is tissue‐specific, the end result being the tissue dependent production of different combinations of peptides from the same precursor. These peptides have a diverse range of biological roles ranging from pigmentation to adrenal function to the regulation of feeding. This level of complexity has resulted in POMC becoming the archetypal model for prohormone processing, illustrating how a single protein combined with post‐translational modification can have a diverse number of roles.

The platelet‐surface thiol isomerase enzyme ERp57 modulates platelet function

Summary.  Background: Thiol isomerases are a family of endoplasmic reticulum enzymes which orchestrate redox‐based modifications of protein disulphide bonds. Previous studies have identified important roles for the thiol isomerases PDI and ERp5 in the regulation of normal platelet function. Aim: Recently, we demonstrated the presence of a further five thiol isomerases at the platelet surface. In this report we aim to report the role of one of these enzymes – ERp57 in the regulation of platelet function. Methods/Results: Using enzyme activity function blocking antibodies, we demonstrate a role for ERp57 in platelet aggregation, dense granule secretion, fibrinogen binding, calcium mobilisation and thrombus formation under arterial conditions. In addition to the effects of ERp57 on isolated platelets, we observe the presence of ERp57 in the developing thrombus in vivo. Furthermore the inhibition of ERp57 function was found to reduce laser‐injury induced arterial thrombus formation in a murine model of thrombosis. Conclusions: These data suggest that ERp57 is important for normal platelet function and opens up the possibility that the regulation of platelet function by a range of cell surface thiol isomerases may represent a broad paradigm for the regulation of haemostasis and thrombosis.

Agouti Related Protein in the Rat Adrenal Cortex: Implications for Novel Autocrine Mechanisms Modulating the Actions of Pro‐Opiomelanocortin Peptides

Agouti related protein (AgRP) is a recently discovered melanocortin receptors (MCR) antagonist implicated in the control of feeding behaviour. Expression of AgRP has been shown to be localized by in situ hybridization to the arcuate nucleus and median eminence of the brain, where it acts as an antagonist to the MC3 and MC4 receptors, while in the periphery the only significant expression was located in the adrenal medulla. As AgRP is only a weak antagonist of the MC2 and MC5 receptors, which are expressed principally by adipocytes and in the adrenal cortex, the question arizes as to the function of peripheral AgRP. In this study, we investigated the expression of AgRP in the rat adrenal and suggest that it is expressed in the adrenal cortex and not as previously described in the medulla. We also show that AgRP mRNA expression is upregulated in the adrenal during fasting and in the contralateral gland following unilateral adrenalectomy but not during chronic stress. These results indicate an as yet undefined role for AgRP in the periphery and are supportive of the suggestion that a further melanocortin receptor exists.

Ovarian expression of insulin-like peptide 3 (INSL3) and its receptor (RXFP2) during development of bovine antral follicles and corpora lutea and measurement of circulating INSL3 levels during synchronized estrous cycles.

Insulin-like peptide 3 (INSL3), a major product of testicular Leydig cells, is also expressed by the ovary, but its functional role remains poorly understood. Here, we quantified expression of INSL3 and its receptor RXFP2 in theca interna cell (TIC) and granulosa cell compartments of developing bovine antral follicles and in corpora lutea (CL). INSL3 and RXFP2 mRNA levels were much higher in TIC than granulosa cell and increased progressively during follicle maturation with INSL3 peaking in large (11-18 mm) estrogen-active follicles and RXFP2 peaking in 9- to 10-mm follicles before declining in larger (11-18 mm) follicles. Expression of both INSL3 and RXFP2 in CL was much lower than in TIC. In situ hybridization and immunohistochemistry confirmed abundant expression of INSL3 mRNA and protein in TIC. These observations indicate follicular TIC rather than CL as the primary site of both INSL3 production and action, implying a predominantly autocrine/paracrine role in TIC. To corroborate the above findings, we showed that in vitro exposure of TIC to a luteinizing concentration of LH greatly attenuated expression of both INSL3 and its receptor while increasing progesterone secretion and expression of STAR and CYP11A1. Moreover, in vivo, a significant cyclic variation in plasma INSL3 was observed during synchronized estrous cycles. INSL3 and estradiol-17β followed a similar pattern, both increasing after luteolysis, before falling sharply after the LH surge. Thus, theca-derived INSL3, likely from the dominant preovulatory follicle, is detectable in peripheral blood of cattle, and expression is down-regulated during luteinization induced by the preovulatory LH surge. Collectively, these findings underscore the likely role of INSL3 as an important intrafollicular modulator of TIC function/steroidogenesis, while raising doubts about its potential contribution to CL function.

Role of gamma-MSH peptides in the regulation of adrenal steroidogenesis.

Alpha-, beta- and gamma-melanocyte stimulating hormones (MSHs) are peptides derived from the ACTH precursor, pro-opiomelanocortin. All three peptides have been highly conserved throughout evolution but their exact biological function in mammals is still largely obscure. In recent years, there has been a surge of interest in alpha-MSH and its role in the regulation of feeding. Gamma-MSH by contrast has been shown to be involved in the regulation of adrenal steroidogenesis and also has effects on the cardiovascular and renal systems. This review will provide an overview of the role that gamma-MSH peptides play in the regulation of adrenal steroidogenesis.

Hox gene expression in adult tissues with particular reference to the adrenal gland.

In comparison to the embryo, very little work has been carried out on the expression and role of Hox genes in the adult animal. An expression profile of all 39 vertebrate Hox genes on a select panel of adult human tissues reveals that in fact these genes are widely expressed throughout the adult human and a colinear pattern of expression is displayed similar to that of the developing embryo. Of particular interest is the abundance of Hox genes that are expressed within the adult adrenal gland. Adrenal cortical cells are continuously renewed to sustain production of zonal steroids. Cell proliferation occurs at the periphery of the cortex and cells are then displaced centripetally, phenotypically switching as they migrate through the gland before undergoing apoptosis at the zona reticularis/medullary boundary. It is still unclear which mechanisms cause the cells to differentiate as they cross the zonal boundaries and we hypothesise that Hox genes may be involved in the phenotypic switching of the adrenocortical cells. In situ hybridisation experiments were carried out on adult rat adrenal gland sections and Hox gene expression was localized within the zonal borders, coinciding with the localization of cells that undergo phenotypic differentiation, and thus supporting our hypothesis that Hox genes may be involved in the phenotypic switching of the adrenocortical cells. As in the developing embryo, the genes display colinear expression with the 3′ Hox genes being expressed within the outer gland and the 5′ genes within the inner zones.

Pro-Opiomelanocortin and adrenal function

Pro-opiomelanocortin (POMC) is a polyhormone precursor produced predominantly in the pars distalis and pars intermedia of the pituitary gland where it undergoes tissue specific processing to produce a whole array of peptides. We have shown previously that peptides derived from the N-terminal region of POMC are involved in adrenal growth in rats. Using specific two site immunoradiometric assays we have found that the plasma of 17 week old fetal sheep contain a 50 fold excess of pro-gamma-MSH over ACTH. As term approached, the levels of pro-gamma-MSH fell and ACTH rose with evidence of fragmentation of pro-gamma-MSH, suggesting that these peptides act in concert in the development of the fetal adrenal cortex and also provide the necessary drive to bring about parturition. In an attempt to explore the pathophysiology of adrenal function we have cloned human POMC cDNA which led to the discovery of a 9bp addition/deletion mutation in the C-terminus of gamma 3-MSH between positions 67-73. Chinese hamster ovary cells (CHO) cells stably transfected with constructs containing the variant POMC cDNAs have shown a degree of partial processing. Work is currently underway to further investigate the effects of these mutations on the processing by the prohormone converting enzymes PC1 and PC2.

Evidence of a possible role for Lys-γ3-MSH in the regulation of adipocyte function

Lys-γ3-MSH is a melanocortin peptide derived from the C-terminal of the 16 kDa fragment of POMC. The physiological role of Lys-γ3-MSH is unclear, although it has previously been shown that, although not directly steroidogenic, it can act to potentiate the steroidogenic response of adrenal cortical cells to ACTH. This synergistic effect appears to be correlated with an ability to increase the activity of hormone sensitive lipase (HSL) and therefore the rate of cholesterol ester hydrolysis. Ligand binding studies have suggested that high-affinity binding sites for Lys-γ3-MSH exist in the adrenal gland and a number of other rat tissues that express HSL, including adipose, skeletal muscle and testes. To investigate the hypothesis that Lys-γ3-MSH may play a wider role in cholesterol and lipid metabolism, we tested the effect of Lys-γ3-MSH on lipolysis, an HSL-mediated process, in 3T3-L1 adipocytes. In comparison with other melanocortin peptides, Lys-γ3-MSH was found to be a potent stimulator of lipolysis. It was also able to phosphorylate HSL at key serine residues and stimulate the hyperphosphorylation of perilipin A. The receptor through which the lipolytic actions of Lys-γ3-MSH are being mediated is not clear. Attempts to characterise this receptor suggest that either the pharmacology of the melanocortin receptor 5 in 3T3-L1 adipocytes is different from that described when expressed in heterologous systems or the possibility that a further, as yet uncharacterised, receptor exists.

The anti-epileptic drug valproic acid (VPA) inhibits steroidogenesis in bovine theca and granulosa cells in vitro.

Valproic acid (VPA) is used widely to treat epilepsy and bipolar disorder. Women undergoing VPA treatment reportedly have an increased incidence of polycystic ovarian syndrome (PCOS)-like symptoms including hyperandrogenism and oligo- or amenorrhoea. To investigate potential direct effects of VPA on ovarian steroidogenesis we used primary bovine theca (TC) and granulosa (GC) cells maintained under conditions that preserve their ‘follicular’ phenotype. Effects of VPA (7.8–500 µg/ml) on TC were tested with/without LH. Effects of VPA on GC were tested with/without FSH or IGF analogue. VPA reduced (P<0.0001) both basal (70% suppression; IC50 67±10 µg/ml) and LH-induced (93% suppression; IC50 58±10 µg/ml) androstenedione secretion by TC. VPA reduced CYP17A1 mRNA abundance (>99% decrease; P<0.0001) with lesser effects on LHR, STAR, CYP11A1 and HSD3B1 mRNA (<90% decrease; P<0.05). VPA only reduced TC progesterone secretion induced by the highest (luteinizing) LH dose tested; TC number was unaffected by VPA. At higher concentrations (125–500 µg/ml) VPA inhibited basal, FSH- and IGF-stimulated estradiol secretion (P<0.0001) by GC without affecting progesterone secretion or cell number. VPA reversed FSH-induced upregulation of CYP19A1 and HSD17B1 mRNA abundance (P<0.001). The potent histone deacetylase (HDAC) inhibitors trichostatin A and scriptaid also suppressed TC androstenedione secretion and granulosal cell oestrogen secretion suggesting that the action of VPA reflects its HDAC inhibitory properties. In conclusion, these findings refute the hypothesis that VPA has a direct stimulatory action on TC androgen output. On the contrary, VPA inhibits both LH-dependent androgen production and FSH/IGF-dependent estradiol production in this in vitro bovine model, likely by inhibition of HDAC.