Age-dependent effects of estradiol on temporal memory: A role for the type 1 cannabinoid receptor?

This study investigated in male mice how age modulates the effects of acute 17ß-estradiol (E2) on dorsal CA1 (dCA1)-dependent retention of temporal associations, which are critical for declarative memory. E2 was systemically injected to young (3-4 months old) and aged (22-24 months old) adult mice either (i) 1 h before the acquisition of an auditory trace fear conditioning (TFC) procedure allowing the assessment of temporal memory retention 24 h later or (ii) during in vivo electrophysiological recordings of CA3 to dCA1 synaptic efficacy under anesthesia. In young mice, E2 induced parallel dose-dependent reductions in memory and synaptic efficacy, i.e. an impairment in TFC retention and a long-term (NMDA receptor-dependent) depression of dCA1 synaptic efficacy as assessed by field excitatory postsynaptic potentials. In contrast, E2 tended to improved TFC retention whilst failing to change synaptic efficacy in aged mice. Age-dependent effects of E2 treatment were confirmed by immunohistochemical analyses of TFC acquisition-elicited dCA1 Fos activation. Thus, such an activation was respectively reduced and enhanced in young and aged E2-treated mice, compared to vehicle treatments. Hippocampal mRNA expression of estrogen receptors by RT-PCR analyses revealed an age-related increase in each receptor mRNA expression. In keeping with the key role of the endocannabinoid system in memory processes and CA3 to dCA1 synaptic plasticity, we next examined the role of cannabinoid type 1 receptors (CB1-R) in the aforementioned age-dependent effects of E2. Having confirmed that mRNA expression of CB1-R diminishes with age, we then observed that the deleterious effects of E2 on both memory and synaptic efficacy were both prevented by the CB1-R antagonist Rimonabant whilst being absent in CB1-R knock out mice. This study (i) reveals age-dependent effects of acute E2 on temporal memory and CA3 to dCA1 synaptic efficacy and (ii) suggests a key role of CB1-R in mediating E2 deleterious effects in young adulthood. Aging-related reductions in CB1-R might thus underlie E2 paradoxical effects across age.

Derivatization-free LC-MS/MS method for estrogen quantification in mouse brain highlights a local metabolic regulation after oral versus subcutaneous administration.

17ß-Estradiol (17ß-E2) is a steroid with pleiotropic actions. In addition to being a sexual hormone, it is also produced in the brain where it modulates the reproductive axis. It has been shown that 17ß-E2 also acts on synaptic plasticity and plays a role in neurological pathways and in neurodegenerative diseases. Assaying this steroid in the brain is thus interesting to improve our knowledge of 17ß-E2 effects in the brain. However, 17ß-E2 concentration in the central nervous system has been reported to be of a few nanograms per gram wet weight (nanomolar range concentration); therefore, its quantification requires both an efficient extraction process and a sensitive detection method. Herein is presented a derivatization-free procedure based on solid-phase extraction followed by LC-MS/MS analysis, targeted on 17ß-E2, its isomer17α-E2, and its metabolites estrone (E1) and estriol (E3). This extraction process allowed reaching 96% 17ß-E2 recovery from the mouse brain. Limit of detection (LOD) and limit of quantification (LOQ) values of 0.5 and 2.5 pmol mL-1, respectively, were reached for both 17α-E2 and 17ß-E2. LOD values for E1 and E3 were 0.01 and 0.025 pmol mL-1, respectively. The variation coefficients for intra- and inter-assays were 6 and 14%, respectively, for both estradiol forms. The method was applied to assess estrogen levels in the mouse brain and hippocampus after 17ß-E2 acute (subcutaneous injection) and chronic (drinking water) physiological administration. Total estrogen levels were determined after enzymatic deconjugation and compared to free estrogen levels. While 17α-E2 was not detected in biological samples, 17ß-E2 and metabolite measurements highlight a local biotransformation of estrogens after physiological administration via drinking water. Graphical abstract Method workflow: After oral or subcutaneous Estradiol administration, mouse brain or hippocampus was removed. Samples were homogenized and prepared according to a liquid-liquid extraction, followed by a solid-phase extraction. Then, LC-MS/MS was optimized to quantify 17ß-E2, its isomer17α-E2, its metabolites estrone (E1) and estriol (E3) and their conjugates.

Estradiol enhances retention but not organization of hippocampus-dependent memory in intact male mice.

Because estrogens have mostly been studied in gonadectomized females, effects of chronic exposure to environmental estrogens in the general population are underestimated. Estrogens can enhance hippocampus-dependent memory through the modulation of information storage. However, declarative memory, the hippocampus-dependent memory of facts and events, demands more than abilities to retain information. Specifically, memory of repetitive events of everyday life such as "where I parked" requires abilities to organize/update memories to prevent proactive interference from similar memories of previous "parking events". Whether such organizational processes are estrogen-sensitive is unknown. We here studied, in intact young and aged adult mice, drinking-water (1µM) estradiol effects on both retention and organizational components of hippocampus-dependent memory, using a radial-maze task of everyday-like memory. Demand on retention vs organization was manipulated by varying the time-interval separating repetitions of similar events. Estradiol increased performance in young and aged mice under minimized organizational demand, but failed to improve the age-associated memory impairment and diminished performance in young mice under high organizational demand. In fact, estradiol prolonged mnemonic retention of successive events without improving organization abilities, hence resulted in more proactive interference from irrelevant memories. c-Fos imaging of testing-induced brain activations showed that the deterioration of young memory was associated with dentate gyrus dysconnectivity, reminiscent of that seen in aged mice. Our findings support the view that estradiol is promnesic but also reveal that such property can paradoxically impair memory. These findings have important outcomes regarding health issues relative to the impact of environmental estrogens in the general population.

Temporal Memory and Its Enhancement by Estradiol Requires Surface Dynamics of Hippocampal CA1 N-Methyl-D-Aspartate Receptors.

BACKGROUND: Identifying the underlying cellular mechanisms of episodic memory is an important challenge, since this memory, based on temporal and contextual associations among events, undergoes preferential degradation in aging and various neuropsychiatric disorders. Memory storage of temporal and contextual associations is known to rely on hippocampal N-methyl-D-aspartate receptor (NMDAR)-dependent synaptic plasticity, which depends ex vivo on dynamic organization of surface NMDARs. Whether NMDAR surface trafficking sustains the formation of associative memory, however, remains unknown. METHODS: We tested this hypothesis, using single nanoparticle imaging, electrophysiology, and behavioral approaches, in hippocampal networks challenged with a potent modulator of NMDAR-dependent synaptic plasticity and memory, 17ß-estradiol (E2). RESULTS: We demonstrate that E2 modulates NMDAR surface trafficking, a necessary condition for E2-induced potentiation at hippocampal cornu ammonis 1 synapses. Strikingly, cornu ammonis 1 NMDAR surface trafficking controls basal and E2-enhanced mnemonic retention of temporal, but not contextual, associations. CONCLUSIONS: NMDAR surface trafficking and its modulation by the sex hormone E2 is a cellular mechanism critical for a major component of episodic memory, opening a new and noncanonical research avenue in the physiopathology of cognition.

Comparative effects of R- and S-equol and implication of transactivation functions (AF-1 and AF-2) in estrogen receptor-induced transcriptional activity.

Equol, one of the main metabolites of daidzein, is a chiral compound with pleiotropic effects on cellular signaling. This property may induce activation/inhibition of the estrogen receptors (ER) a or b, and therefore, explain the beneficial/deleterious effects of equol on estrogen-dependent diseases. With its asymmetric centre at position C-3, equol can exist in two enantiomeric forms (R- and S-equol). To elucidate the yet unclear mechanisms of ER activation/inhibition by equol, we performed a comprehensive analysis of ERa and ERb transactivation by racemic equol, as well as by enantiomerically pure forms. Racemic equol was prepared by catalytic hydrogenation from daidzein and separated into enantiomers by chiral HPLC. The configuration assignment was performed by optical rotatory power measurements. The ER-induced transactivation by R- and S-equol (0.1-10 µM) and 17b-estradiol (E2, 10 nM) was studied using transient transfections of ERα and ERß in CHO, HepG2 and HeLa cell lines. R- and S-equol induce ER transactivation in an opposite fashion according to the cellular context. R-equol and S-equol are more potent in inducing ERα in an AF-2 and AF-1 permissive cell line, respectively. Involvement of ERα transactivation functions (AF-1 and AF-2) in these effects has been examined. Both AF-1 and AF-2 are involved in racemic equol, R-equol and S-equol induced ERα transcriptional activity. These results could be of interest to find a specific ligand modulating ER transactivation and could contribute to explaining the diversity of equol actions in vivo.

Respective contribution exerted by AF-1 and AF-2 transactivation functions in estrogen receptor alpha induced transcriptional activity by isoflavones and equol: consequence on breast cancer cell proliferation.

Estrogens used in hormone replacement therapy regimens may increase the risk of developing breast cancer. Paradoxically, high consumption of plant-derived phytoestrogens, particularly soybean isoflavones, is associated with a low incidence of breast cancer. To explore the molecular basis for these potentially different experimental/clinical outcomes, we investigated whether soybean isoflavones elicit distinct transcriptional actions from estrogens by performing transient transfections in different cell lines. Our results demonstrate that 17beta estradiol (E2), isoflavones, and equol (EQ) effectively trigger the transcriptional activation with both estrogen receptors (ER), ER alpha and ER beta. ER alpha transcriptional activity is mediated through two transactivation domains AF-1 and AF-2, whose activity is tightly regulated in a cell-type and promoter-specific manner. Isoflavones, genistein, and daidzein (DAI), and EQ, the main estrogenic metabolite of DAI, are ER alpha agonists for transcriptional activation. The molecular mechanisms for ER alpha-induced transcriptional activity by isoflavones and EQ involve their capacity to act mainly through AF-1 regardless of the cell type. Therefore, our data indicate that estrogenic ligands, such as isoflavones and EQ, exert their effects on ER alpha transactivation similarly to the endogenous ligand E2, and suggest that the risk of estrogen-related diseases might not be reduced by soy-rich food and/or isoflavone- or EQ-based supplements.

Enterodiol and enterolactone, two major diet-derived polyphenol metabolites have different impact on ERalpha transcriptional activation in human breast cancer cells.

Lignans are plant compounds metabolized in the mammalian gut to produce the estrogenic enterolignans, enterodiol (ED) and enterolactone (EL). Because estrogens have been linked to breast cancer etiology, enterolignans could affect breast cancer risk, but to our knowledge, the mechanisms by which they exert their estrogenic and/or anti-estrogenic effects in humans are still unclear. To better understand how estrogenic compounds from the food, such as the enterolignans, might influence breast cancer progression and their mechanisms to interfere with human estrogen receptor (ER) signalling in hormone-dependant diseases, we examined and compared the ability of ED, EL and 17beta-estradiol (E2) to induce the transactivation of ERalpha and ERbeta, to modulate ERalpha target genes, to exert either growth stimulatory or anti-proliferative effects and finally to modulate MCF-7 cell migration by acting on matrix metalloproteases (MMP)-2 and -9, at concentrations that are achievable through a lignan-rich diet. This study indicates that enterolignans show distinct properties for transactivation of ERalpha and ERbeta. ED, as E2, induces ERalpha transcriptional activation through transactivation functions AF-1 and AF-2, while EL is less efficient in inducing AF-1, acting predominantly through AF-2. Furthermore, ED and EL modulate ERalpha mRNA and protein contents as well as MCF-7 cell proliferation and secreted MMP activities in a different way. Enterolignans are compounds of wide interest nowadays and our results help to unveil their mechanisms of action on ER, emphasizing the fact that the dietary load in lignans could be of importance in the balance between being risk or chemopreventive factors for breast cancer and women's health.

Activation of the estrogen receptor contributes to the progression of pulmonary lymphangioleiomyomatosis via matrix metalloproteinase-induced cell invasiveness.

CONTEXT: The role of estrogens in the pathogenesis of lymphangioleiomyomatosis (LAM), an aggressive and destructive, eventually fatal lung disease of women, is poorly understood. OBJECTIVE: The study was conducted to test the hypothesis that the lung disease in LAM is estrogen mediated and to determine whether estrogens contribute to the invasiveness of LAM. DESIGN: In vitro cell culture of spindle-shaped LAM cells (LAMD-SM) were isolated and propagated from affected lungs. Estrogen receptor (ER)-alpha and ERbeta analyses were conducted by RT-PCR. ERalpha and ERbeta, tissue inhibitor of metalloproteinase-2, and matrix metalloproteinases (MMP)-2 had Western blot analysis for protein assessment. Activity assays were performed for MT1-MMP, MMP-2, and tissue inhibitor of metalloproteinase-2. Assessment of MMP-2 promoter function was done via transfection assays. Cell invasion chambers were used to determine and quantitate cell invasiveness. SETTING: The study was conducted at an academic medical center. PATIENTS: Tissue and cells were obtained from patients as outlined in approved institution review board protocol (97/007). INTERVENTION: LAMD-SM cells were treated with a specific MMP-2 antibody or a nonspecific inhibitor, doxycycline. MAIN OUTCOME MEASURES: Activity of MMP-2 and invasiveness of LAMD-SM cells were measured. RESULTS: LAMD-SM cells express functional ERs (ERalpha and ERbeta), which undergo rapid intracellular turnover in their unbound state. 17beta-estradiol (E(2)) enhances the transcriptional ER activity. E(2)-induced ER activation increases synthesis and activity of MMP-2 through posttranscriptional mechanisms in LAMD-SM. The E(2)/ER-mediated increase of MMP-2 promotes LAMD-SM invasiveness, in assays in vitro, which can be inhibited by specific antibodies against MMP-2 or doxycycline, an inhibitor of MMPs. CONCLUSION: The invasion and destruction of lung parenchyma in LAM is, at least partially, an estrogen-MMP-driven process, which has major implications for therapeutic interventions.

Glomerular aging in females is a multi-stage reversible process mediated by phenotypic changes in progenitors.

The glomeruli of postmenopausal C57BL6 mice, and age-matched males, show progressive hypertrophy and glomerulosclerosis. We asked whether this was a multistage process, was due to alterations in glomerular progenitors, and was reversible in female mice. Using cross bone marrow transplants (BMT) between young and old females, we found that BMT delivered a phenotype that was donor age-specific. The fact that lesions in young recipients were more severe if the donors were in late rather than early menopause suggested that new progenitor phenotypes had appeared. Postmenopausal recipients of BMT from young donors had reduced glomerular hypertrophy and sclerosis, implying that the aging lesions in females were reversible and that progenitors, rather than the local environment, determined the glomerular profile. The altered phenotype included increased extracellular matrix synthesis and decreased matrix metalloproteinase-2 levels as well as cell hypertrophy. The mechanism of the cellular hypertrophy was due to uncoupling of hypertrophy from proliferation, resulting from elevated p27 levels. Thus, glomerular hypertrophy and sclerosis in aging females is a multistage process, is reversible, and may be determined by the phenotype of bone marrow-derived progenitor cells.

Autocrine activation of the local insulin-like growth factor I system is up-regulated by estrogen receptor (ER)-independent estrogen actions and accounts for decreased ER expression in type 2 diabetic mesangial cells.

Autocrine activation of the IGF-I system in mesangial cells (MC) promotes glomerular scarring in a model of type 1 diabetes. Although estrogens protect against progressive nondiabetic glomerulosclerosis (GS), women with diabetes seem to loose the estrogen-mediated protection against cardiovascular disease. However, little is known about the local IGF-I system and its interactions with estrogens in the pathogenesis of type 2 diabetic GS. Therefore, we examined db/db B6 (db/db) mice, a model of type 2 diabetes and diabetic GS. The IGF-I system was activated in the glomeruli and MC of female diabetic db/db mice, but not in nondiabetic db/+ littermates. We found increased IGF-I receptor (IGFR) expression and activation, including activation of MAPK. Surprisingly, estrogens, via an estrogen receptor (ER)-independent mechanism(s), increased IGFR expression, IGFR and insulin receptor substrate phosphorylation, and extracellular signal-regulated kinase activation in db/db MC. In contrast, ER expression was decreased in MC and glomeruli of db/db mice. Treatment with a neutralizing antibody to IGF-I or the MAPK inhibitor PD98059 increased ER expression and transcriptional activity. This suggests that the local prosclerotic IGF-I system is activated in type 2 diabetes and diminishes ER-mediated protection against GS. Although estrogens may stimulate protective ER signaling, they also activate the IGF-I system via ER-independent mechanisms in db/db MC. The later estrogen effects appear to outweigh the antisclerotic effects of ER activation. This may in part account for loss of estrogen protection against the progression of diabetic GS in women with type 2 diabetes.

The glomerulosclerosis of aging in females: contribution of the proinflammatory mesangial cell phenotype to macrophage infiltration.

Age-associated renal changes may be an important cause of renal failure. We recently found that aged female B6 mice developed progressive glomerular lesions. This was associated with macrophage infiltration, a frequent finding in glomerulosclerosis. We used these mice as a model for studying the mechanisms of glomerular aging. We compared the gene expression profile of intact glomeruli from late postmenopausal (28-month-old) mice to that of intact glomeruli from premenopausal (5-month-old) mice. We found that inflammation-related genes, especially those expressed by activated macrophages, were up-regulated in the glomeruli of 28-month-old mice, a result correlating with the histological observation of glomerular macrophage infiltration. The mechanism for macrophage recruitment could have been stable phenotypic changes in mesangial cells because we found that mesangial cells isolated from 28-month-old mice expressed higher levels of RANTES and VCAM-1 than cells from 5-month-old mice. The elevated serum tumor necrosis factor (TNF)-alpha levels present in aged mice may contribute to increased RANTES and VCAM-1 expression in mesangial cells. Furthermore, cells from 28-month-old mice were more sensitive to TNF-alpha-induced RANTES and VCAM-1 up-regulation. The effect of TNF-alpha on RANTES expression was mediated by TNF receptor 1. Interestingly, mesangial cells isolated from 28-month-old mice had increased nuclear factor-kappaB transcriptional activity. Inhibition of nuclear factor-kappaB activity decreased baseline as well as TNF-alpha-induced RANTES and VCAM-1 expression in mesangial cells isolated from 28-month-old mice. Thus, phenotypic changes in mesangial cells may predispose them to inflammatory stimuli, such as TNF-alpha, which would contribute to glomerular macrophage infiltration and inflammatory lesions in aging.

Development of albuminuria and glomerular lesions in normoglycemic B6 recipients of db/db mice bone marrow: the role of mesangial cell progenitors.

The pathologic hallmarks of diabetic nephropathy are excess mesangial extracellular matrix (ECM) and mesangial cell proliferation. We previously showed that mesangial cell phenotypic changes play an important role in the pathogenesis of diabetic nephropathy. We concluded that phenotypic changes were present in bone marrow (BM)-derived mesangial cell progenitors, as transplantation of BM from db/db mice, a model of type 2 diabetic nephropathy, transferred the db genotype and a nephropathy phenotype to naive B6 mice recipients. The recipients did not develop diabetes; however, they did develop albuminuria and glomerular lesions mirroring those in the donors (i.e., glomerular hypertrophy, increased ECM, and increased cell number with cell proliferation). We found that matrix metalloproteinase 2 (MMP-2) facilitated invasion of the mesangial cells into ECM and proliferation in vitro. Thus, increased MMP-2 activity in db/db mesangial cell progenitors may partially explain increased mesangial cell repopulation and proliferation in B6 recipients of db/db BM. In summary, BM-derived mesangial cell progenitors may play a crucial role in the development and progression of ECM accumulation and mesangial cell proliferation in this model of diabetic nephropathy in type 2 diabetes.

Response to sex hormones differs in atherosclerosis-susceptible and -resistant mice.

Genetic factors that determine the degree of susceptibility to atherosclerosis may also influence the effects of estrogens and progestins in arterial vessel disease. We examined and compared estrogen receptor (ER) and progesterone receptor (PR) expression and the effects of 17beta-estradiol (E2) and progesterone (P) on collagen synthesis and matrix metalloproteinase (MMP) activities in the aortic arch and in cultured aortic smooth muscle cells (ASMC) of atherosclerosis-susceptible (C57Bl6/J, B6) or -resistant (C3H/HeJ, C3H) mice. ERalpha, ERbeta, and PR levels were higher in the aorta and ASMC of atherosclerosis-susceptible B6 mice. In transfection studies using an estrogen response element-driven reporter plasmid, E2 elicited a >2-fold increase in luciferase activity in ASMC of B6 (B6-ASMC), which demonstrated the transcriptional activity of ER in atherosclerosis-susceptible cells. Importantly, the response of endogenous target genes to E2 and P was different in B6-ASMC and C3H-ASMC. E2 decreased collagen synthesis but had no effect on MMP activities in B6-ASMC. P decreased MMP-2 and MMP-9 activity in B6-ASMC. In contrast, E2 increased MMP-2 and decreased MMP-9 activity but had no effect on collagen synthesis in C3H-ASMC. P had no effect on collagen synthesis and MMP activity in C3H-ASMC. These differences in response to sex hormones may have important implications for women who receive hormone replacement therapy.

Estrogen deficiency accelerates progression of glomerulosclerosis in susceptible mice.

Estrogen deficiency may contribute to the development and progression of glomerulosclerosis in postmenopausal women. The responsiveness to estrogens could be controlled by genetic traits related to those that determine the susceptibility to glomerular scarring. This study was undertaken to determine whether the intensity of the sclerotic response was modified by the estrogen status in sclerosis-prone ROP Os/+ mice. Ovariectomized ROP Os/+ mice developed more severe renal dysfunction and glomerulosclerosis than intact, ie, estrogen sufficient age-matched female mice. Ovariectomized ROP Os/+ exhibited increased accumulation of extracellular matrix, predominantly of laminin, and a marked distortion of the glomerular architecture. We found an increase in macrophage infiltration in the mesangium of ovariectomized ROP Os/+. Estrogen deficiency decreased glomerular estrogen receptor expression in ROP Os/+ mice, which we had previously found to be low in the parental ROP strain. Thus, although physiological estrogen levels in young ROP Os/+ mice could not prevent the development of glomerulosclerosis, estrogen deficiency accelerated the progression of glomerular scarring in this mouse strain. This suggests that estrogen replacement will slow but not prevent the progression of glomerulosclerosis. It underscores the importance of the genetic composition of individuals that determines the susceptibility to diseases as well as the response to treatment.

Estrogen-related abnormalities in glomerulosclerosis-prone mice: reduced mesangial cell estrogen receptor expression and prosclerotic response to estrogens.

The development and progression of glomerulosclerosis (GS) is determined by the genetic background. The incidence of end-stage renal disease is increased in postmenopausal women, suggesting that estrogen deficiency may play a role in the accumulation of extracellular matrix by mesangial cells (MCs), which are primarily responsible for the synthesis and degradation of this matrix. Using mouse models that are prone or resistant to the development of GS, we compared the expression of estrogen receptor (ER)-alpha and ER-beta subtypes in GS-prone and GS-resistant glomeruli and isolated MCs, and examined the effects of estrogens on ER, collagen, and matrix metalloproteinase (MMP) expression in MCs. Glomeruli and MCs from GS-prone mice had decreased expression of ER-alpha and ER-beta subtypes and ER transcriptional activity was also decreased in their MCs. Importantly, although 17 beta-estradiol treatment resulted in decreased collagen accumulation and increased MMP-9 expression and activity in MCs from GS-resistant mice, there was, paradoxically, no effect on collagen accumulation and decreased MMP-9 expression and activity in MCs from GS-prone mice. Thus, GS susceptibility is associated with diminished ER expression in MCs. The renal protective effects of estrogens, including decreased collagen accumulation and increased MMP-9 expression, seem to be blunted in GS-prone MCs.

Expression and regulation of estrogen receptors in mesangial cells: influence on matrix metalloproteinase-9.

Diabetic glomerulosclerosis is characterized by the accumulation of extracellular matrix (ECM) in the mesangium. Estrogens seem to retard whereas estrogen deficiency seems to accelerate progressive glomerulosclerosis. Thus, mesangial cells (MC) may be a target for estrogens. Estrogen action is mediated via estrogen receptor (ER) subtypes ERalpha and ERbeta. Both ER subtypes were expressed in human and mouse MC. Using an estrogen-responsive reporter construct in transfection assays, it also was demonstrated that the nuclear ER were transcriptionally active. In the presence of 17beta-estradiol (E2; 10(-10) to 10(-8) M), there was a progressive increase in the mRNA levels of both ERalpha (approximately 1.8-fold and approximately 2.7-fold after 24 and 72 h, respectively) and ERbeta (approximately 1.3-fold and approximately 2.2-fold after 24 and 72 h, respectively). ERalpha protein levels increased approximately 2.5-fold after 24 h (10(-10) M, E2) and up to approximately 5.4-fold after 72 h (10(-9) M, E2). ERbeta protein levels increased approximately 2.1-fold in the presence of E(2) (10(-9) M) after 24 h. Thus, estrogens positively regulate the expression of the ER subtypes, thereby maintaining or increasing MC responsiveness to estrogens. Because diabetic glomerulosclerosis may be due partly to a decrease in ECM degradation, the effects of estrogens on matrix metalloproteinases (MMP) were studied. It was found that E2 (10(-10) to 10(-8) M) increased both MMP-9 mRNA and MMP-9 activity in MC. This may be an important mechanism by which estrogens influence ECM turnover and protect against progression of diabetic glomerulosclerosis.