Presence Of Glucocorticoids Research Articles

Aromatase regulation and breast cancer.

Aromatase regulation and breast cancer.

High constitutive glucocorticoid receptor beta in human neutrophils enables them to reduce their spontaneous rate of cell death in response to corticosteroids.

Neutrophils are markedly less sensitive to glucocorticoids than T cells, making it difficult to control inflammation in neutrophil-mediated diseases. Development of new antiinflammatory strategies for such diseases would be aided by an understanding of mechanisms underlying differential steroid responsiveness. Two protein isoforms of the human glucocorticoid receptor (GR) exist, GRalpha and GRbeta, which arise from alternative splicing of the GR pre-mRNA primary transcripts. GRbeta does not bind glucocorticoids and is an inhibitor of GRalpha activity. Relative amounts of these two GRs can therefore determine the level of glucocorticoid sensitivity. In this study, human neutrophils and peripheral blood mononuclear cells (PBMCs) were studied to determine the relative amounts of each GR isoform. The mean fluorescence intensity (MFI) using immunofluorescence analysis for GRalpha was 475 +/- 62 and 985 +/- 107 for PBMCs and neutrophils, respectively. For GRbeta, the MFI was 350 +/- 60 and 1,389 +/- 143 for PBMCs and neutrophils, respectively (P < 0.05). After interleukin (IL)-8 stimulation of neutrophils, there was a statistically significant increase in intensity of GRbeta staining to 2,497 +/- 140 (P < 0.05). No change in GRalpha expression was observed. This inversion of the GRalpha/GRbeta ratio in human neutrophils compared with PBMCs was confirmed by quantitative Western analysis. Increased GRbeta mRNA expression in neutrophils at baseline, and after IL-8 exposure, was observed using RNA dot blot analysis. Increased levels of GRalpha/GRbeta heterodimers were found in neutrophils as compared with PBMCs using coimmunoprecipitation/Western analysis. Transfection of mouse neutrophils, which do not contain GRbeta, resulted in a significant reduction in the rate of cell death when treated with dexamethasone.We conclude that high constitutive expression of GRbeta by human neutrophils may provide a mechanism by which these cells escape glucocorticoid-induced cell death. Moreover, upregulation of this GR by proinflammatory cytokines such as IL-8 further enhances their survival in the presence of glucocorticoids during inflammation.

Hormones increase mRNA of cyclic-nucleotide-gated cation channels in airway epithelia

Previous studies have shown that the mRNA of cyclic-nucleotide-gated nonselective cation (CNG) channels is expressed in rat airway epithelia and that these channels contribute to sodium-mediated short-circuit currents in cultured rat tracheal epithelia. Patch-clamp studies from human A549 cells indicate that these channels contribute to cGMP-stimulated L-cis-diltiazem- and dichlorobenzamil-inhibited whole-cell sodium currents. This study demonstrates that mRNA for primary and secondary subunits of CNG channels, halphaCNG1 and hbetaCNG1 respectively, are expressed in several human airway cell lines, including normal and cystic fibrosis bronchial airway cells, in normal and cystic fibrosis tracheal airway cell lines and nasal polyp tissue from a cystic fibrosis patient. The mRNA of ralphaCNG1 in rat lung increased in response to increased circulating glucocorticoids and decreased in animals with lowered circulating glucocorticoids after aminoglutethimide treatment. Likewise the mRNA of halphaCNG1 increased in the presence of glucocorticoids in cultured alveolar airway cells. The mRNA of alphaCNG1 in rat lung was also increased in response to a low-salt diet and lowered in animals fed a high-salt diet. Likewise the mRNA of alphaCNG1 was increased in response to increased aldosterone and decreased in animals given spironolactone. These results suggest that mRNA for alphaCNG1 increases in response to elevated glucocorticoids or mineralocorticoids. Because alphaCNG1 is a functional sodium entry channel in both rat and human airway epithelial cells, if channel protein is also elevated this channel could mediate an increase in sodium absorption across lung epithelia in response to circulating hormones.

Glucocorticoids maintain the extracellular matrix of differentiated mammary tissue during explant and whole organ culture.

Mouse mammary whole organ culture (WOC) and explant culture of lactating tissue were used to investigate the mechanism by which glucocorticoids maintain secretory epithelium following lobuloalveolar development. The relative number of mammary epithelial cells expressing glucocorticoid receptors did not change with the loss of secretory epithelium during involution as demonstrated with competitive binding assays and immunohistochemistry for the glucocorticoid receptor. Furthermore, glucocorticoids did not inhibit AP-1 binding activity. However, Northern analysis demonstrated that genes associated with the breakdown of the extracellular matrix were not expressed in tissues cultured with glucocorticoids, in contrast to their upregulation during involution of mammary tissue cultured with insulin alone. Tissue inhibitor of metalloproteinase-1 (TIMP-1) mRNA expression was lowest in tissue cultured in the presence of glucocorticoids and increased 2.3-, 3.4-, and 9-fold when tissues were involuted in the presence of insulin (Ins) alone, Ins and hydrocortisone (Hyd) with 0. 005 mg/ml, or 0.01 mg/ml collagenase IV, respectively. These data indicate that glucocorticoids maintain mammary differentiation in part by inhibiting the turnover of basement membrane.

Induction of resistance to active experimental allergic encephalomyelitis by myelin basic protein-specific Th2 cell lines generated in the presence of glucocorticoids and IL-4

We have produced T cell lines with a Th2 phenotype in the presence of IL-4 and the glucocorticoid dexamethasone (DEX). IL-4 and DEX together were more effective in inducing a Th2 response than IL-4 alone. Myelin basic protein (MBP)-specific Th2 lines were obtained and their ability to induce experimental allergic encephalomyelitis (EAE) was studied. Lines treated with IL-4 and DEX did not transfer passive EAE and did not induce cellular infiltration into the central nervous system as opposed to the encephalitogenic Th1 lines. Lines treated with IL-4 and DEX did not protect animals from the effect of encephalitogenic Th1 lines when the two were injected together. However, a high proportion of animals injected with IL-4 + DEX-treated lines became refractory to the development of EAE after immunization with MBP; that is, it was possible to induce resistance to active EAE without prior episodes of disease. Interestingly, animals injected with T cell lines had accelerated antibody responses against MBP and the predominant isotype was dependent on the cytokines synthesized by the T cell line injected. There was not evidence that the resistance to active EAE was due to anergy of MBP-reactive cells or the action of CD8+ cells. Our data suggest that MBP-specific T cell lines prevent the induction of disease by deviating the reactivity to MBP from a cell-mediated to a humoral one and not merely from a Th1 to a Th2 response.

Induction of resistance to active experimental allergic encephalomyelitis by myelin basic protein-specific Th2 cell lines generated in the presence of glucocorticoids and IL-4

Induction of resistance to active experimental allergic encephalomyelitis by myelin basic protein-specific Th2 cell lines generated in the presence of glucocorticoids and IL-4

Glucocorticoid-induced contractility and F-actin content of human lung fibroblasts in three-dimensional culture.

Fibroblast contractility plays a useful role in the wound healing process but contributes to architectural distortion in the lungs. Glucocorticoids (GCs) have been reported to reduce dermal fibroblast contractility, which may result in delaying wound healing, but the effects on lung fibroblasts are unknown. In this study, we examined how human lung fibroblast contractility is altered in the presence of GCs. Lung fibroblast cell lines (n = 5) were established from normal parts of surgically resected lung tissue. The effects of GCs on contractility were investigated with a type I collagen gel contraction assay. Filamentous actin (F-actin) content was detected by confocal microscopy and measured with a fluorescent phalloidin binding assay. GCs augmented fibroblast contraction in a concentration-dependent manner, with an approximate EC(50) of 1.8 x 10(-8) M, whereas other steroid derivatives had no effects. GC contractility needed de novo protein synthesis. The GC-induced increase in contractility was found to be consistent with an increase in F-actin content. In conclusion, lung fibroblast contractility was enhanced with GCs through an upregulation of lung fibroblast F-actin.

Thymosin beta 4 sulfoxide is an anti-inflammatory agent generated by monocytes in the presence of glucocorticoids.

The possibility that glucocorticoids upregulate the expression of anti-inflammatory mediators is an exciting prospect for therapy in inflammatory diseases, because these molecules could give the therapeutic benefits of steroids without toxic side effects. Supernatants from monocytes and macrophages cultured in the presence of glucocorticoids increase the dispersion of neutrophils from a cell pellet in the capillary tube migration assay. This supernatant factor, unlike other neutrophil agonists, promotes dispersive locomotion of neutrophils at uniform concentration, lowers their adhesion to endothelial cells, inhibits their chemotactic response to fMLP and induces distinctive morphological changes. Here we show that thymosin beta4 sulfoxide is generated by monocytes in the presence of glucocorticoids and acts as a signal to inhibit an inflammatory response. In vitro, thymosin beta4 sulfoxide inhibited neutrophil chemotaxis, and in vivo, the oxidized peptide, but not the native form, was a potent inhibitor of carrageenin-induced edema in the mouse paw. Thymosin beta4 is unique, because oxidation attenuates its intracellular G-actin sequestering activity, but greatly enhances its extracellular signaling properties. This description of methionine oxidation conferring extracellular function on a cytosolic protein may have far-reaching implications for future strategies of anti-inflammatory therapy.

HIV-1 long terminal repeat modulation by glucocorticoids in monocytic and lymphocytic cell lines

Glucocorticoid gene regulation can be carried out through direct binding of glucocorticoid receptor to glucocorticoid responsive elements (GRE), regulating directly gene transcription and modulating some signaling pathways. The human immunodeficiency virus type 1 (HIV-1) expression can be activated by different immunomodulators through binding of particular nuclear factors to its long terminal repeat (LTR). In order to investigate the effect of glucocorticoids in pathways that activate HIV-1 expression, we transfected promonocyte (U937) and T lymphocyte (CEM-T4) cell lineages with a plasmid containing the chloramphenicol acetyl transferase (CAT) reporter gene under the control of the HIV-1 LTR. In U937 cells, dexamethasone (DEX) downregulates CAT expression induced by either phorbol myristate acetate (PMA), tumor necrosis factor alpha (TNFα) or granulocyte/macrophage-colony stimulating factor (GM-CSF). In CEM-T4 cells the CAT activity was slightly upregulated by DEX following the induction by either PMA or TNFα. Interestingly, in both cell lines transactivation of this reporter gene by transactivator protein (TAT) was downregulated by DEX. When the CAT gene was under control of HIV-1 enhancer isolated from its LTR background, the CAT activity induced by PMA was not affected by the presence of glucocorticoids. In all experiments, comparable data were obtained when DEX was replaced by hydrocortisone (HC). Our results show that, depending on the cell line, glucocorticoids can differently affect HIV-1 expression, probably by interfering in cellular pathways involved in virus expression. Moreover, the target of this regulation in LTR is probably not the enhancer region itself.

Regulation of major histocompatibility complex class II antigens on human alveolar macrophages by granulocyte-macrophage colony-stimulating factor in the presence of glucocorticoids.

Alveolar macrophages (AM) present antigen poorly to CD4+ T cells and respond weakly to interferon-gamma (IFN-gamma) for up-regulation of major histocompatibility complex (MHC) class II and costimulatory molecule expression. In atopic asthma, however, AM exhibit enhanced antigen-presenting cell (APC) activity. Since granulocyte-macrophage colony-stimulating factor (GM-CSF) is increased in the airways of asthmatic patients, we have investigated its role in modulating the APC function of AM. The effects of glucocorticoids were also studied since earlier studies showed optimal induction of MHC antigens on monocytes by GM-CSF in their presence. GM-CSF in the presence, but not the absence, of dexamethasone enhanced the expression of HLA-DR, -DP and -DQ antigens by AM. However AM and monocytes differed in the optimal concentration of steroid required to mediate this effect (10-10 m and 10-7 m, respectively). Induction of MHC antigens was glucocorticoid specific and independent of IFN-gamma. These studies suggest the existence of an IFN-gamma-independent pathway of macrophage activation, which may be important in regulating APC function within the lung.

Hormonal regulation and cellular localization of fatty acid synthase in human fetal lung.

Fatty acid synthase (FAS; EC 2.3.1.85) supplies de novo fatty acids for pulmonary surfactant synthesis, and FAS gene expression is both developmentally and hormonally regulated in the fetal lung. To further examine hormonal regulation of FAS mRNA and to determine the cellular localization of FAS gene expression, we cultured human fetal lungs (18-22 wk gestation) as explants for 1-4 days in the absence (control) or presence of glucocorticoid [dexamethasone (Dex), 10 nM] and/or cAMP agents (8-bromo-cAMP, 0.1 mM and IBMX, 0.1 mM). FAS protein content and activity increased similarly in the presence of Dex (109 and 83%, respectively) or cAMP (87 and 111%, respectively), and responses were additive in the presence of both hormones (230 and 203%, respectively). With a rabbit anti-rat FAS antibody, FAS immunoreactivity was not detected in preculture lung specimens but appeared in epithelial cells lining the tubules with time in culture. Dex and/or cAMP markedly increased staining of epithelial cells, identified as type II cells, whereas staining of mesenchymal fibroblasts was very low under all conditions. With in situ hybridization, FAS mRNA was found to be enriched in epithelial cells lining the alveolar spaces, and the reaction product increased in these cells when the explants were cultured with the hormones. The increased FAS mRNA content in the presence of Dex and/or cAMP is primarily due to increased stabilization of mRNA, although Dex alone increased the transcription rate by approximately 30%. We conclude that hormonal treatment of cultured human fetal lungs increases FAS gene expression primarily by increasing stability of the message. The induction of FAS during explant culture and by hormones occurs selectively in type II epithelial cells, consistent with the regulatory role of this enzyme in de novo synthesis of fatty acid substrate for surfactant synthesis in perinatal lungs.

Glucose-6-phosphatase mRNA levels in kidney isolated tubule suspensions are increased by dexamethasone and decreased by insulin

The strong induction of renal glucose-6-phosphatase (G6Pase) during starvation has been suggested to be responsible for the increased role of the kidney in glucose production during long-term fasting. To investigate whether this induction may be caused by a direct hormonal effect on the renal proximal tubular cell, we incubated rat renal tubule suspensions in the presence of glucocorticoids or insulin for 6 hours; normoxia was required, since hypoxic conditions were associated with markedly decreased G6Pase mRNA levels despite maintenance of adenosine triphosphate (ATP) levels. The G6Pase mRNA level was increased twofold to threefold by 10 −8 to 10 −5 mol/L dexamethasone (DXM), whereas the most effective concentration of insulin, 10 −9 mol/L, induced only a 40% decrease. These results suggest that the increased role of the kidney in glucose production during long-term starvation could be linked to a direct effect of glucocorticoids on renal G6Pase.

Transcriptional control of the murine polymeric IgA receptor promoter by glucocorticoids.

Glucocorticoids have been implicated as an important regulator of intestinal epithelial cell ontogeny. The polymeric IgA receptor (pIgR) is expressed in the intestinal epithelial layer and is regulated by several mediators, including glucocorticoids. The mechanism of how corticosteroids alter the transcriptional regulation of pIgR expression has not been defined. In this study, we demonstrated that glucocorticoids upregulate steady-state pIgR mRNA levels in the proximal intestine of suckling rats and in the IEC-6 intestinal cell line. We performed functional analysis of the 5'-flanking region in the presence of glucocorticoids and its receptor using the intestinal cell line Caco-2. We screened 4.7 kb of the upstream region of the murine gene and identified the most potent steroid response element to reside between nt -215 and -163 relative to the start of transcription. Substitution mutation analysis of this region identified the location of the putative steroid response element to be between nt -195 and -176. In vitro DNase I footprint analysis using the recombinant glucocorticoid receptor DNA binding domain confirmed a single area of protection that spans the nt identified by mutagenesis analysis. Electrophoretic mobility shift assays of the putative element confirmed the binding of both recombinant and cell synthesized glucocorticoid receptor in a specific manner. In summary, we report the identification and characterization of the glucocorticoid-DNA response element located in the immediate 5'-upstream region of the murine pIgR gene.

Insulin-Like Growth Factor (IGF) Binding Proteins Modulate the Glucocorticoid-Dependent Biological Effects of IGF-II in Cultured Fetal Rat Hepatocytes

The role of insulin-like growth factor binding proteins (IGFBPs) in regulation by IGF-II of glycogenesis and DNA synthesis was investigated in hepatocytes isolated from fetal rat livers at days 15 and 18 of gestation and grown in the presence or absence of cortisol. IGFBP-1 was clearly revealed by Western ligand blot and immunoblot analysis of IGFBPs secreted into conditioned media. Its production and cellular messenger RNA (mRNA) were positively regulated by cortisol and increased in older cells. In the absence of IGFBP (fresh medium), glycogenesis, and DNA synthesis were stimulated by IGF-II and insulin. In each case, cortisol enhanced this stimulation. In the presence of IGFBPs (cell-conditioned media), IGF-II stimulation of DNA synthesis and to a lesser extent glycogenesis was inhibited. The degree of inhibition was directly related to IGFBP-1 production. IGFBPs had no effect on stimulation of glycogenesis and DNA synthesis by des(1–6)IGF-II, a structural analog of IGF-II that does not bind to IGFBPs. Insulin, whose biological effects were not modified by conditioned media, inhibited IGFBP-1 production. Comparison of the dose dependence of the two bioactivities showed that DNA synthesis was more sensitive to IGF-II than glycogenesis. Our results suggest that in the case of DNA synthesis the effects of IGF-II are mediated via the IGF-I receptor and those of insulin via the insulin receptor, whereas in the case of glycogenesis both are mediated via the insulin receptor. In conclusion, IGF-II and insulin stimulation of glycogenesis and DNA synthesis in cultured fetal hepatocytes depends on the presence of glucocorticoid and the stage of development. IGF-II action is negatively regulated by IGFBP-1 whose synthesis increases in the presence of glucocorticoids.

Insulin-like growth factor (IGF) binding proteins modulate the glucocorticoid-dependent biological effects of IGF-II in cultured fetal rat hepatocytes.

The role of insulin-like growth factor binding proteins (IGFBPs) in regulation by IGF-II of glycogenesis and DNA synthesis was investigated in hepatocytes isolated from fetal rat livers at days 15 and 18 of gestation and grown in the presence or absence of cortisol. IGFBP-1 was clearly revealed by Western ligand blot and immunoblot analysis of IGFBPs secreted into conditioned media. Its production and cellular messenger RNA (mRNA) were positively regulated by cortisol and increased in older cells. In the absence of IGFBP (fresh medium), glycogenesis, and DNA synthesis were stimulated by IGF-II and insulin. In each case, cortisol enhanced this stimulation. In the presence of IGFBPs (cell-conditioned media), IGF-II stimulation of DNA synthesis and to a lesser extent glycogenesis was inhibited. The degree of inhibition was directly related to IGFBP-1 production. IGFBPs had no effect on stimulation of glycogenesis and DNA synthesis by des(1-6)IGF-II, a structural analog of IGF-II that does not bind to IGFBPs. Insulin, whose biological effects were not modified by conditioned media, inhibited IGFBP-1 production. Comparison of the dose dependence of the two bioactivities showed that DNA synthesis was more sensitive to IGF-II than glycogenesis. Our results suggest that in the case of DNA synthesis the effects of IGF-II are mediated via the IGF-I receptor and those of insulin via the insulin receptor, whereas in the case of glycogenesis both are mediated via the insulin receptor. In conclusion, IGF-II and insulin stimulation of glycogenesis and DNA synthesis in cultured fetal hepatocytes depends on the presence of glucocorticoid and the stage of development. IGF-II action is negatively regulated by IGFBP-1 whose synthesis increases in the presence of glucocorticoids.

Gap junctional communication and regulation of the glycogenic response to insulin by cell density and glucocorticoids in cultured fetal rat hepatocytes.

Cell culture studies have revealed that metabolic functions of the adult hepatocyte are related to cell density. Development of the glycogenic response to insulin under glucocorticoid control was investigated in 15- and 18-day-old fetal rat hepatocytes plated at different cell densities. After culturing for 48 hours with glucocorticoids, the stimulatory effect of insulin on [14C]glucose incorporation into glycogen after 3 hours progressed from weak response (less than 1.4-fold) in sparse cultures to a maximal response in dense ones (3.0- to 4.5-fold), depending on the fetal stage. The response was always no more than 2.0-fold in the absence of glucocorticoids, even with dense cultures. Such a dual regulation pattern was not found for the glycogenolytic effect of glucagon similarly expressed regardless of culture conditions. When cells were clustered in limited circular regions of the dish, the insulin response was higher than for sparse cultures for a similar number of cells per culture. Using the scrape-loading technique with Lucifer Yellow CH, a positive dye transfer was obtained in clustered cultures providing that they were grown in the presence of glucocorticoids; insulin as well as glucagon stimulated twofold intercellular communication. Connexin32 (Cx32) and connexin26 (Cx26) protein levels were assayed by Western immunoblotting and developed according to age and exposure to glucocorticoids. Thus, glucocorticoids through development of gap junctions enabled establishment of intercellular communication that could be stimulated by insulin and glucagon in cultured fetal hepatocytes. Gap junction functioning and the biologic effect of insulin correlated closely.

Long-term lack of endogenous glucocorticoids down-regulates glucocorticoid receptor levels in the rat forebrain

To understand the effect of a chronic lack of endogenous glucocorticoids on glucocorticoid receptor levels, the changes of glucocorticoid receptor content in the rat forebrain five months after adrenalectomy were investigated. In the long-term adrenalectomized rats that showed a hormone deficiency and loss of glucocorticoid receptor immunoreactivity in the forebrain, an intraperitoneal injection of corticosterone was used to elevate the serum hormone levels and recover glucocorticoid receptor immunoreactivity in the forebrain. One hour later, when the blood corticosterone returned to the normal level, the recovery of glucocorticoid receptor immunoreactivity in the forebrain was examined by immunohistochemistry. Since the complete restoration of glucocorticoid receptor immunoreactivity was shown to depend on the presence of normal levels of both serum hormone and intracellular glucocorticoid receptors, the weak reappearance of glucocorticoid receptor immunoreactivity in any forebrain area of the long-term adrenalectomized rats that had normal serum corticosterone might reflect the low intracellular glucocorticoid receptor levels there. Our results revealed a weak reappearance of glucocorticoid receptor immunoreactivity in some forebrain areas of the long-term adrenalectomized rats after corticosterone treatment; the hippocampal granule cell layer and cerebral cortex in particular showed very weak recovery of glucocorticoid receptor immunoreactivity. Conversely, neurons in the CA1/CA2 subfields of the hippocampal pyramidal cell layer, immediately adjacent to the granule cell layer on the same brain section, exhibited a strong reappearance of glucocorticoid receptor immunoreactivity, to near normal levels. These results suggest that, five months after adrenalectomy, the intracellular glucocorticoid receptor content decreased in the rat granule cell layer and cerebral cortex. Therefore, the long-term lack of endogenous glucocorticoids after adrenalectomy might down-regulate but not up-regulate the intracellular glucocorticoid receptor level, and the presence of glucocorticoids is important for the continued synthesis of glucocorticoid receptors.

Growth Hormone and Dexamethasone Stimulate Lipolysis and Activate Adenylyl Cyclase in Rat Adipocytes by Selectively Shifting Gi 2 to Lower Density Membrane Fractions

GH, in the presence of glucocorticoid, produces a delayed increase in lipolysis in rat adipose tissue, but the biochemical mechanisms that account for this action have not been established. Other lipolytic agents rapidly activate adenylyl cyclase (AC) and the resulting production of cAMP initiates a chain of reactions that culminates in the activation of hormone-sensitive lipase. We compared responses of segments of rat epididymal fat or isolated adipocytes to 30 ng/ml GH and 0.1 μg/ml dexamethasone (Dex) with 0.1 ng/ml isoproterenol (ISO), which evoked a similar increase in lipolysis. All measurements were made during the fourth hour after the addition of GH+Dex or immediately after the addition of ISO to cells or tissues that had been preincubated for 3 h without hormone. Although no significant increases in cAMP were discernible in homogenates of GH+Dex-treated tissues, RP-cAMPS (RP-adenosine 3′5′-phosphothioate), a competitive inhibitor of cAMP, was equally effective in decreasing lipolysis induced by GH+Dex or ISO. The proportion of PKA that was present in the active form was determined by measuring the incorporation of 32P from[γ -32P]ATP into kemptide in the absence and presence of saturating amounts of cAMP. GH+Dex and ISO produced similar increases in protein kinase A activity in tissue extracts. Treatment with GH+Dex did not change the total forskolin-stimulated AC present in either a crude membrane pellet sedimented at 16K × g or a less dense membrane pellet sedimented at 100K × g, but doubled the AC activity in the 16K pellet when assayed in the absence of forskolin. To evaluate possible effects on G proteins, pellets obtained from centrifugation of adipocyte homogenates at 16K × g and 100K × g were solubilized and subjected to PAGE and Western analysis. GH+Dex decreased Giα2 by 44% (P < 0.02) in the 16K pellets and increased it by 52% (P < 0.01) in the 100K pellets. Gsα in the 16K pellet was unaffected by GH+Dex and was decreased (P < 0.05) in the 100K pellet. Sucrose density fractionation of the 16K pellets revealed a similar GH+Dex-dependent shift of Giα2 to less dense fractions as determined by both Western analysis and[ 32P]NAD ribosylation catalyzed by pertussis toxin. No such changes were seen in the distribution of Gsα or 5′-nucleotidase. Colchicine (100 μm) blocked the GH+Dex-dependent shift of Giα2 from the 16K to the 100K pellet and blocked the lipolytic effects of GH+Dex, but not those of ISO. We conclude that by modifying the relationship between AC and Giα2, GH+Dex relieves some inhibition of cAMP production and consequently increases lipolysis.

Growth hormone and dexamethasone stimulate lipolysis and activate adenylyl cyclase in rat adipocytes by selectively shifting Gi alpha2 to lower density membrane fractions.

GH, in the presence of glucocorticoid, produces a delayed increase in lipolysis in rat adipose tissue, but the biochemical mechanisms that account for this action have not been established. Other lipolytic agents rapidly activate adenylyl cyclase (AC) and the resulting production of cAMP initiates a chain of reactions that culminates in the activation of hormone-sensitive lipase. We compared responses of segments of rat epididymal fat or isolated adipocytes to 30 ng/ml GH and 0.1 microg/ml dexamethasone (Dex) with 0.1 ng/ml isoproterenol (ISO), which evoked a similar increase in lipolysis. All measurements were made during the fourth hour after the addition of GH+Dex or immediately after the addition of ISO to cells or tissues that had been preincubated for 3 h without hormone. Although no significant increases in cAMP were discernible in homogenates of GH+Dex-treated tissues, Rp-cAMPS (Rp-adenosine 3'5'-phosphothioate), a competitive inhibitor of cAMP, was equally effective in decreasing lipolysis induced by GH+Dex or ISO. The proportion of PKA that was present in the active form was determined by measuring the incorporation of 32P from [gamma-32P]ATP into kemptide in the absence and presence of saturating amounts of cAMP. GH+Dex and ISO produced similar increases in protein kinase A activity in tissue extracts. Treatment with GH+Dex did not change the total forskolin-stimulated AC present in either a crude membrane pellet sedimented at 16K x g or a less dense membrane pellet sedimented at 100K x g, but doubled the AC activity in the 16K pellet when assayed in the absence of forskolin. To evaluate possible effects on G proteins, pellets obtained from centrifugation of adipocyte homogenates at 16K x g and 100K x g were solubilized and subjected to PAGE and Western analysis. GH+Dex decreased Gi alpha2 by 44% (P < 0.02) in the 16K pellets and increased it by 52% (P < 0.01) in the 100K pellets. Gs alpha in the 16K pellet was unaffected by GH+Dex and was decreased (P < 0.05) in the 100K pellet. Sucrose density fractionation of the 16K pellets revealed a similar GH+Dex-dependent shift of Gi alpha2 to less dense fractions as determined by both Western analysis and [32P]NAD ribosylation catalyzed by pertussis toxin. No such changes were seen in the distribution of Gs alpha or 5'-nucleotidase. Colchicine (100 microM) blocked the GH+Dex-dependent shift of Gi alpha2 from the 16K to the 100K pellet and blocked the lipolytic effects of GH+Dex, but not those of ISO. We conclude that by modifying the relationship between AC and Gi alpha2, GH+Dex relieves some inhibition of cAMP production and consequently increases lipolysis.

Basic fibroblast growth factor in the presence of dexamethasone stimulates colony formation, expansion, and osteoblastic differentiation by rat bone marrow stromal cells.

Basic fibroblast growth factor (bFGF) is known to stimulate endosteal bone formation in vivo by a mechanism possibly mediated via osteoblast precursor cells present in the bone marrow. In high density cultures of primary bone marrow cells, and in the presence of glucocorticoids, bFGF stimulates the formation of a bone-like matrix; however, due to the dense nature of these cultures, the exact mechanism of action is unclear. In an adaptation of the fibroblastic colony formation unit assay, in which the bone marrow cells are grown in the presence of dexamethasone, beta-glycerophosphate, and ascorbate, mineralized colonies are formed which stem from single mesenchymal precursor cells and grow in isolation from each other. Using this system we have been able to investigate the mechanism by which bFGF stimulates the formation of bone like tissue in vitro. We have shown that bFGF increases the formation of a calcified collagenous matrix in vitro by (1) increasing the total number of fibroblastic colonies formed, (2) increasing the proportion of differentiated colonies that synthesize collagen and calcify, and (3) stimulating the proliferation and collagen accumulation of the individual colonies. A maximal increase in total and differentiated colony numbers was seen after only 5 days exposure to bFGF, however, continued exposure to bFGF continued to increase the size and collagen content of the individual colonies. Bearing in mind the endosteal location of newly formed bone seen after treatment with bFGF, these processes may well play an active role in this effect.