Megalin and androgen receptor gene expression in young and old human skeletal muscle before and after three sequential exercise bouts

Androgen signaling occurs primarily via the androgen receptor. Megalin, a low-density lipoprotein endocytic receptor located in various mammalian tissues, has been recently shown to facilitate sex hormone–binding globulin (SHBG) steroid complexes across cell membranes. The purpose of this investigation is to determine if the megalin gene is expressed in human skeletal muscle and if present to determine how megalin and androgen receptor mRNA expression change in response to sequential exercise bouts with respect to aging. Ten younger (age: 18-25 years) and 10 older (age: 60-75 years) men completed 3 workouts (M, W, F) each consisting of 9 sets of lower-body exercises with 10 repetitions per set at 80% 1 repetition maximum. Vastus lateralis muscle biopsies were extracted at baseline (T1), 48 hours after workout 1 (T2) and 2 (T3), and 24 hours after workout 3 (T4), and blood samples were collected before and 5 minutes after each workout. Muscle was analyzed for megalin and androgen receptor expression using gene-specific primers and SYBR green chemistry, and blood was analyzed for serum testosterone, SHBG, and the free androgen index. Megalin was expressed in both young and old subjects across all time points, although no between- or within-group mean differences were detected at any time point. Androgen receptor was expressed higher in young men at all time points compared to in old men (p < 0.05), and a significant correlation (p < 0.05; r = 0.506) was found between serum testosterone and androgen receptor after workout 1. Based on our data, the gene coding for megalin is expressed inside skeletal muscle, but its role, if any, in steroid cellular transport cannot be determined. This finding could lay the groundwork for more mechanistic investigations to better delineate its functional role and its potential as a therapeutic adjunct for androgen-related disorders in healthy and aged populations.     

Structure/function analyses of human sex hormone-binding globulin: effects of zinc on steroid-binding specificity

In humans, sex hormone-binding globulin (SHBG) binds and transports the biologically most important androgens and estrogens in the blood, and regulates the access of these steroids to their targets tissues. In addition to binding sex steroids, SHBG has specific binding sites for divalent cations including calcium and zinc. Zinc binding to a site at the entrance of the steroid-binding pocket in human SHBG has been shown to reduce its affinity for estrogens, while having no impact on the binding of C19 steroids. Crystallographic studies indicate that C18 and C19 steroids are bound in opposite orientations within the SHBG steroid-binding site, and we have obtained new information that supports a molecular model explaining the mechanism by which zinc alters the affinity of human SHBG for estrogens, by studying directly the estradiol-binding properties SHBG variants created by site-directed mutagenesis. In this model, the coordination of a zinc ion by the side chains of residues Asp65 and His136 eliminates a critical hydrogen bond between Asp65 and the hydroxyl at C3 of estrogens, such as estradiol and 2-methoxyestradiol, and causes disorder in a polypeptide loop segment that covers the steroid-binding site. The combination of these structural changes explains the specific decrease in the affinity of human SHBG for C18 steroids in the presence of a zinc ion.     

Co-expression and interaction of cubilin and megalin in the adult male rat reproductive system

Cubilin is a peripheral membrane protein that cooperates with the endocytic receptor megalin to mediate endocytosis of ligands in various polarized epithelia. Megalin is expressed in the male reproductive tract where it has been implicated in the process of sperm membrane remodeling. A potential role for cubilin in the male reproductive tract has not been explored. Using RT-PCR, we found that cubilin and megalin mRNAs are expressed in the efferent ducts, corpus and cauda epididymis, and proximal and distal vas deferens. Immunohistological analysis revealed that cubilin was expressed in nonciliated cells of the efferent ducts, principal cells of the corpus and cauda epididymis and vas deferens. Immunogold EM showed cubilin in endocytic pits, endocytic vesicles, and endosomes of these cells. The expression profile of cubilin in the male reproductive tract was coincident with that of megalin except in principal cells of the caput epididymis. Double immunogold labeling showed that cubilin and megalin co-localized within the endocytic apparatus and recycling vesicles of efferent duct cells. Neither protein was found in lysosomes. Injection of RAP, an antagonist of megalin interaction with cubilin, reduced the level of intracellular cubilin in cells of the efferent ducts and vas deferens. In conclusion, cubilin and megalin are co-expressed in cells of the epididymis and vas deferens and the endocytosis of cubilin in these tissues is dependent on megalin. Together, these findings highlight the potential for a joint endocytic role for cubilin and megalin in the male reproductive tract.     

Estrogen and androgen regulation of sex hormone binding globulin secretion by a human liver cell line

Both estrogens and androgens have been shown to stimulate sex hormone binding globulin (SHBG) secretion in vitro in the hepatocellular carcinoma cell line, Hep G2, in contrast to the expected inhibition by androgens from in vivo studies. However, such in vitro stimulation was only demonstrated at high steroid doses, generally in serum-containing medium, with added Phenol Red. In the present study, Hep G2 cells were grown in serum-free medium, without Phenol Red, under the influence of testosterone (T) (0, 0.5-500 nM) and ethinyl estradiol (EE2) (0, 50 pM-500 nM). Levels of secreted SHBG and albumin were correlated with androgen receptors in cytosolic (ARc) and nuclear (ARn) fractions and with DNA levels. In the presence of increasing T levels, SHBG levels fell to 39% of control values at 5 nM T (P = 0.047), rising to 97% of control at 500 nM. Conversely, incubation with EE2 produced a rise in SHBG secretion of more than 100% at 0.5 nM (P less than 0.02) which was sustained to 50 nM (P less than 0.005). DNA levels did not change with the addition of testosterone or EE2, with the exception of a 15% reduction at 5 nM EE2 (P less than 0.05). Albumin levels in the medium were not significantly altered by either steroid. However, in response to T, androgen receptor (AR) levels were reduced in cytosolic (42% of control) and nuclear (22%) fractions at 5 nM, and these changes in ARc and ARn correlated with SHBG levels over the range of T concentrations (P = 0.04 and P = 0.017, respectively). Nuclear estrogen receptor (ER) increased over 10-fold at 5 and 50 pM EE2 (P less than 0.001) and maintained 50 nM (P less than 0.001). Cytosolic ER was reduced at 0.5 and 5 nM but recovered at 50 nM, correlating with SHBG levels (P less than 0.001). These findings are consistent with the hypothesis that estrogens and androgens regulate SHBG synthesis in man by direct, specific, probably receptor-mediated effects on hepatocytes. Hep G2 cells grown in serum-free medium are a suitable experimental system for further study of this phenomenon.     

Sex steroid hormone binding globulin levels and free 17 beta-estradiol and testosterone in cynomolgus monkeys during different reproductive states

The effect of differences in sex hormone binding globulin (SHBG) levels associated with reproductive status on free 17 beta-estradiol (E2) and testosterone (T) was characterized in cynomolgus monkeys. Cycling female cynomolgus monkeys had higher SHBG levels than males, pregnant and lactating females (P less than 0.05). Ovariectomized females were not different than control females, suggesting no hypoestrogenic effect. The percentage of free T was elevated in pregnant animals (P less than 0.05) compared to normal males and females, but the percentage of free E2 was similar between these groups. Although a gender difference in the percentages of free E2 and T was not detected, there was a gender difference in the free T and E2 concentrations due to endogenous secretion. Increased free E2 concentrations during pregnancy were the result of endogenous secretion rather than the decreased binding capacity of SHBG; the increased percentage of free T during pregnancy significantly increased free T concentrations. These data suggest that the gender difference in SHBG levels in cynomolgus monkeys is due to androgenic influences and that estrogens have minimal influence. Furthermore, the decrease in SHBG levels during pregnancy and lactation may not be entirely dependent on these androgenic influences.     

A cytoplasmic PPPSP motif determines megalin's phosphorylation and regulates receptor's recycling and surface expression

Megalin is a large endocytic receptor expressed at the apical surface of several absorptive epithelia. It binds multiple ligands including apolipoproteins, vitamin and hormone carrier proteins and signaling molecules such as parathyroid hormone and the morphogen sonic hedgehog. An important characteristic of megalin is its high endocytic activity, which is mediated by tyrosine-based endocytic motifs within the receptor's cytoplasmic tail. This domain also harbors several putative consensus phosphorylation motifs for protein kinase (PK) C and casein kinase-II and one consensus motif for PKA and glycogen synthase kinase-3 (GSK3). Here we report that the cytoplasmic domain of megalin is constitutively phosphorylated depending on the integrity of a PPPSP motif, a putative GSK3 site, with a minor participation of the other phosphorylation motifs. Mutation of the serine residue within the PPPSP motif as well as blocking GSK3 activity, with two different inhibitors, significantly decreased the phosphorylation levels of the receptor. Both the megalin PPPAP mutant and the underphosphorylated wild-type receptor, by inhibition of GSK3 activity, were more expressed at the cell surface and more efficiently recycled, but they were not inhibited in their initial endocytosis rates. Altogether, these results show that the PPPSP motif and the GSK3 activity are critical to allow megalin phosphorylation and also negatively regulate the receptor's recycling.     

Megalin antagonizes activation of the parathyroid hormone receptor

Parathyroid hormone (PTH) is predominantly cleared from the circulation by glomerular filtration and degradation in the renal proximal tubules. Here, we demonstrate that megalin, a multifunctional endocytic receptor in the proximal tubular epithelium, mediates the uptake and degradation of PTH. Megalin was purified from kidney membranes as the major PTH-binding protein and shown in BIAcore analysis to specifically bind full-length PTH and amino-terminal PTH fragments (Kd 0.5 microM). Absence of the receptor in megalin knockout mice resulted in 4-fold increased levels of amino-terminal PTH fragments in the urine. In F9 cells expressing both megalin and the PTH/PTH-related peptide receptor (PTH/PTHrP receptor), uptake and lysosomal degradation of the hormone was mediated through megalin. Blocking megalin-mediated clearance of PTH resulted in 3-fold increased stimulation of the PTH/PTHrP receptor. These data provide evidence that megalin is involved in the renal catabolism of PTH and potentially antagonizes PTH/PTHrP receptor activity in the proximal tubular epithelium.     

Interactions between human plasma sex hormone-binding globulin and xenobiotic ligands

Human sex hormone-binding globulin (SHBG) binds sex steroids with high affinity. In plasma, the number of SHBG steroid-binding sites far exceeds the molar concentrations of sex steroids, and will accommodate other ligands such as phytoestrogens and fatty acids. We have therefore developed a screening assay to identify ligands for SHBG, which exist in our diet or environment. This assay allows the binding of potential ligands to SHBG to be assessed under physiological conditions, and is sensitive to the effects of plasma constituents. Several classes of endocrine active compounds were tested, including hydroxy-polychlorinated biphenyls (HO-PCBs), phthalate esters, monoesters, chlorinated pesticides, as well as synthetic estrogens and phytoestrogens. The relative binding affinities (RBAs) of various compounds to SHBG were determined in competitive displacement assays, by comparison with 17 beta-estradiol (RBA=100). Synthetic estrogens bound SHBG with RBAs of 0.4 (ethinylestradiol)-0.2 (diethylstilbestrol), while some phytoestrogens bound with RBAs of 0.12 (coumestrol)-0.04 (naringenin). Many compounds did not bind to SHBG with sufficient affinity to allow RBA measurements, and these include: several phytoestrogens, such as genistein and kaempferol, polychlorinated biphenyls, phthalate esters and monoesters. Of nine HO-PCB congeners tested only 4-OH-2', 3', 4', 5'-tetraCB and 4-OH-2, 2', 3', 4', 5'-pentaCB bound SHBG in undiluted serum with RBAs of 0.05 and 0.11. Although all test compounds bound to SHBG with much lower affinity than endogenous sex steroids, these interactions may be physiologically relevant in situations where plasma SHBG levels are high and endogenous sex steroid levels are low, such as in pre-pubertal children and women taking oral contraceptives.     

Endocytic trafficking of megalin/RAP complexes: dissociation of the complexes in late endosomes.

Megalin (gp330) is a member of the low-density lipoprotein receptor gene family. Like other members of the family, it is an endocytic receptor that binds a number of specific ligands. Megalin also binds the receptor-associated protein (RAP) that serves as an exocytic traffic chaperone and inhibits ligand binding to the receptor. To investigate the fate of megalin/RAP complexes, we bound RAP glutathione-S-transferase fusion protein (RAP-GST) to megalin at the surface of L2 yolk sac carcinoma cells and followed the trafficking of the complexes by immunofluorescence and immunogold labeling and by their distribution on Percoll gradients. We show that megalin/RAP-GST complexes, which are internalized via clathrin-coated pits, are delivered to early endosomes where they accumulate during an 18 degrees C temperature block and colocalize with transferrin and transferrin receptor. Upon release from the temperature block, the complexes travel to late endosomes where they colocalize with rab7 and can be coprecipitated with anti-RAP-GST antibodies. Dissociation of the complex occurs in late endosomes and is most likely triggered by the low pH (approximately 5.5) of this compartment. RAP is then rapidly delivered to lysosomes and degraded whereas megalin is recycled to the cell surface. When the ligand, lipoprotein lipase, was bound to megalin, the receptor was found to recycle through early endosomes. We conclude that in contrast to receptor/ligand complexes, megalin/RAP complexes traffic through late endosomes, which is a novelty for members of the low-density lipoprotein receptor gene family.     

Megalin functions as an endocytic sonic hedgehog receptor

Embryos deficient in the morphogen Sonic hedgehog (Shh) or the endocytic receptor megalin exhibit common neurodevelopmental abnormalities. Therefore, we have investigated the possibility that a functional relationship exists between the two proteins. During embryonic development, megalin was found to be expressed along the apical surfaces of neuroepithelial cells and was coexpressed with Shh in the ventral floor plate of the neural tube. Using enzyme-linked immunosorbent assay, homologous ligand displacement, and surface plasmon resonance techniques, it was found that the amino-terminal fragment of Shh (N-Shh) bound to megalin with high affinity. Megalin-expressing cells internalized N-Shh through a mechanism that was inhibited by antagonists of megalin, viz. anti-receptor-associated protein and anti-megalin antibodies. Heparin also inhibited N-Shh endocytosis, implicating proteoglycans in the internalization process, as has been described for other megalin ligands. Use of chloroquine to inhibit lysosomal proteinase activity showed that N-Shh endocytosed via megalin was not efficiently targeted to the lysosomes for degradation. The ability of megalin-internalized N-Shh to bypass lysosomes may relate to the finding that the interaction between N-Shh and megalin was resistant to dissociation with low pH. Together, these findings show that megalin is an efficient endocytic receptor for N-Shh. Furthermore, they implicate megalin as a new regulatory component of the Shh signaling pathway.     

Sex Steroid Receptors in the Perinatal Rat Brain

Many sex differences in the regulation of reproductive functionin rats are the result of a differentiation of the brain whichoccurs neonatally. Although injections of either androgens orestrogens are capable during the neonatal period of alteringhypothalamic systems involved in reproductive behavior and gonadotropinregulation, the physiological role of each type of hormone hasnot been clearly established. In both sexes, circulating estrogensare normally kept from interacting with estrogen receptors inthe limbic brain by the high levels of alpha-fetoprotein inthe blood. The local aromatization of androgens in the braincould circumvent alpha-fetoprotein, since androgens do not bindto this serum protein. The "paromatization hypothesis" statesthat testosterone, which is abundant in neonatal male circulationbut absent in females, is locally converted to estradiol inthe limbic brain. There it binds to estrogen receptor proteinsto produce tissue differentiation. The ontogeny of estradiolbinding proteins in limbic areas is consistent with the aromatizationhypothesis, with a rapid increase in receptor levels occurringshortly after birth. Also, the presence of endogenous estradiol-receptorcomplexes has been demonstrated in the cell nuclei of male neonatesbut not female neonales. Furthermore, the presence of estradiolbinding proteins in other regions of the neonatal male and femalebrain suggests an additional role of estradiol. unknown as ofyet. Several studies with agents which block the aromatizationof androgens to estrogens or the binding of estrogens to theirreceptors are consistent with the aromatization hypothesis,since these agents prevent the differentiation of intact neonatalmales. However, specific androgen binding proteins are alsopresent in neonatal brains, and androgen-receptor complexescan be found in cell nuclei of neonates after an injection oftritiated androgen. The possible involvement of these receptorsin sexual differentiation of the brain is suggested by the findingthat an antiandrogen inhibits both the binding of androgens(but not estrogens) and the differentiation of males.     

Estradiol induction of cAMP in breast cancer cells is mediated by foetal calf serum (FCS) and sex hormone-binding globulin (SHBG).

Plasma sex hormone-binding globulin (SHBG or SBP), the specific carrier for estradiol and androgens, after binding to its membrane receptor (SHBG-R), causes a significant increase of cAMP in the presence of estradiol, in both breast (MCF-7) and prostate (LNCaP) cancer cells maintained in serum-free medium. On the other hand, it has been proposed that estrogens, in addition to the well-known nuclear receptor pathway, exert their biological effect inducing cAMP, as a consequence of a direct membrane action, in breast cancer and uterine cells. The aim of the present study was to clarify this controversial issue by verifying if the cAMP increase in MCF-7 cells was a direct effect of estradiol, or if it was mediated by FCS proteins, such as bovine sex hormone-binding globulin; and to reevaluate the effect of human SHBG on cAMP induction in the presence of FCS. MCF-7 cells were maintained in DCC-FCS (treated with DCC to remove steroids), in SHBG-FREE/DCC-FCS (treated with DCC and with a specific affinity chromatography to remove bovine sex hormone-binding globulin), or in serum-free medium (SFM). It was observed that estradiol determined a significant time-dependent increase of cAMP only in MCF-7 cells maintained in 10% DCC-FCS. When cells were maintained in 10% SHBG-FREE/DCC-FCS, estradiol had no detectable effect. However, its ability to increase cAMP was observed again after the addition of human SHBG, in doses ranging from 5 to 50 nM. Moreover, in the presence of 10% SHBG-FREE/DCC-FCS, SHBG, even in the absence of estradiol, caused a significant increase of cAMP. In conclusion, the data reported in the present study suggest that the ability of estradiol to induce cAMP in MCF-7 cells is not due to a direct membrane effect of the hormone, but rather it is mediated by FCS. SHBG is one of the serum factors mediating estradiol action. Lastly, it was proven that SHBG triggers the cAMP pathway in MCF-7 cells in a physiologic culture condition and at physiologic concentrations.     

(Arene)Cl₂Ru(II) complexes with N-coordinated estrogen and androgen isonicotinates: interaction with sex hormone binding globulin and anticancer activity

(Arene)dichloridoruthenium(II) complexes with N-coordinated isonicotinates of androgens (6) and estrogens (9) were prepared and tested for affinity to the estrogen receptor (ERα) and sex hormone binding globulin (SHBG), as well as for cytotoxicity in cancer cells. None of the new complexes bound noticeably to the ER and most of them also bound less strongly to SHBG than the corresponding unmetallated steroids 7. In MTT assays the Ru(p-cymene) complexes 9 of 2-substituted estrones were equally or even more cytotoxic than the metal-free steroids against hormone-dependent (MCF-7 breast and KB-V1 cervix carcinomas) and hormone-independent (518A2 melanoma) cells. The addition of external SHBG to MTT assays lowered the cytotoxicities of the complexes 9 and distinctly more so those of some steroids 7, probably by the way of sequestration and reduction of the cellular uptake. In the absence of SHBG the estrogen complexes 9 were internalized by 518A2 melanoma cells and ruthenated their DNA as quantified by ICP-OES. They also ruthenated salmon sperm DNA but did not change the topology of plasmid DNA in EMSA experiments. In addition, the Ru(p-cymene) complex of 2-ethoxyestrone (9c) was shown to reduce the motility of 518A2 melanoma cells in a wound-healing assay.     

Longitudinal associations of the endocrine environment on fat partitioning in postmenopausal women

Among postmenopausal women, declining estrogen may facilitate fat partitioning from the periphery to the intra-abdominal space. Furthermore, it has been suggested that excess androgens contribute to a central fat distribution pattern in women. The objective of this longitudinal study was to identify independent associations of the hormone milieu with fat distribution in postmenopausal women. Fifty-three healthy postmenopausal women, either using or not using hormone replacement therapy (HRT) were evaluated at baseline and 2 years. The main outcomes were intra-abdominal adipose tissue (IAAT), subcutaneous abdominal adipose tissue, and total thigh fat analyzed by computed tomography scanning and leg fat and total body fat mass measured by dual-energy X-ray absorptiometry. Serum estradiol, estrone, estrone sulfate, total testosterone, free testosterone, androstenedione, dehydroepiandrosterone sulfate), sex hormone-binding globulin (SHBG), and cortisol were assessed. On average, in all women combined, IAAT increased by 10% (10.5 cm(2)) over 2 years (P < 0.05). Among HRT users, estradiol was inversely associated with, and estrone was positively associated with, 2-year gain in IAAT. Among HRT nonusers, free testosterone was inversely associated with, and SHBG was positively associated with, 2-year gain in IAAT. These results suggest that in postmenopausal women using HRT, greater circulating estradiol may play an integral role in limiting lipid deposition to the intra-abdominal cavity, a depot associated with metabolically detrimental attributes. However, a high proportion of weak estrogens may promote fat partitioning to the intra-abdominal cavity over time. Furthermore, among postmenopausal women not using HRT, greater circulating free testosterone may limit IAAT accrual.     

Changes in sex hormone binding globulin, high density lipoprotein cholesterol and plasma lipids in male cyclists during training and competition.

This study was performed on 13 professional race-cyclists to examine changes in sex hormone binding globulin (SHBG), high density lipoprotein cholesterol (HDL-C) and serum lipid concentrations after training and after competition. While SHBG, total cholesterol and phospholipids increased and free fatty acids (FFA) decreased significantly during training, HDL-C and FFA increased and SHBG and triglycerides (TG) decreased significantly during the competition period. These latter changes in serum lipids and lipoproteins were assumed to be a direct effect of utilisation of muscle and plasma TG as fuels for exertion occurring only in extreme exercise. Changes in SHBG concentrations indicated that they were dependent on the conditions of the physical effort and could be related not only to the concentrations of androgens but also to the reduction in body mass.     

Evidence for the role of megalin in renal uptake of transthyretin

The kidney is a major organ for uptake of the thyroid hormone thyroxine (T(4)) and its conversion to the active form, triiodothyronine. In the plasma, one of the T(4) carriers is transthyretin (TTR). In the present study we observed that TTR, the transporter of both T(4) and retinol-binding protein, binds to megalin, the multiligand receptor expressed on the luminal surface of various epithelia including the renal proximal tubules. In the kidney, megalin plays an important role in tubular uptake of macromolecules filtered through the glomerulus. To evaluate the importance of megalin for renal uptake of TTR, we performed binding/uptake assays using immortalized rat yolk sac cells with high expression levels of megalin. Radiolabeled TTR, free as well as in complex with thyroxine or retinol-binding protein, was rapidly taken up by the cells, and the uptake was strongly inhibited by a polyclonal megalin antibody and by the receptor-associated protein, a chaperone-like protein inhibiting ligand binding to megalin. In cell culture, different TTR mutations presented different levels of cell association and degradation, suggesting that the structure of TTR is important for megalin recognition. Both the apo form and the T(4)-bound form were taken up by the cells. Analysis of urine from patients with Dent's disease, a renal tubular disorder that alters receptor-mediated endocytic reabsorption of proteins, identified TTR as an abundant excreted protein. Furthermore, analysis of kidney sections of megalin-deficient mice revealed no immunohistochemical TTR labeling in intracellular vesicles in the proximal tubule cells when compared with wild type control littermates. Taken together, the present data indicate that TTR represents a novel megalin ligand of importance in the thyroid hormone homeostasis.     

Serum androgens and sex hormone binding globulin in obese Pima Indian females

Levels of serum androgens and sex hormone binding globulin (SHBG) were measured in 20 obese Pima Indian females aged 19–44 and compared with those of normal-weight Caucasians aged 20–46. The Pima exhibited significantly decreased SHBG compared to Caucasians, but a strong effect of age on androgen levels rendered mean comparisons useless. Androstenedione (A) and dehydroepiandrosterone-sulfate (DHEA-S) decreased significantly, and testosterone (T) declined slightly with age in the Pima, whereas these androgens showed no significant decreases in Caucasians for this age range. A possible relationship of androgens to the Pima female's propensity for android obesity as well as possible effects of obesity on SHBG, androgens, and aging is discussed.