Estrogen prevents glucocorticoid-induced apoptosis in osteoblasts in vivo and in vitro.

The ability of estrogen to prevent glucocorticoid-induced apoptosis in osteoblasts was studied both in vitro and in vivo. Glucocorticoid treatment for 72 h produced a dose-dependent increase in the number of apoptotic cells, determined by acridine orange/ethidium bromide staining, with a maximal response of 31+/-2% and 26+/-3% with 100 nM corticosterone in primary rat and mouse osteoblasts, respectively. Simultaneous administration of varying concentrations of 17beta-estradiol and 100 nM corticosterone decreased apoptotic osteoblasts in a dose-dependent manner, with a maximal decrease of 70% with 0.01 nM 17beta-estradiol. Terminal deoxynucleotidyltransferase-mediated deoxy-UTP-biotin nick end labeling also demonstrated glucocorticoid-induced DNA fragmentation that was inhibited by estrogen. Estrogen was shown to inhibit apoptosis induced by lipopolysaccharide treatment. As early as 6 h, Western blots demonstrated a dose-dependent decrease in the Bcl-2/Bax ratio, which reached a minimum of 0.18 in osteoblasts treated with 1000 nM corticosterone for 72 h. This reduction in Bcl-2/Bax was abolished by treating osteoblasts simultaneously with 17beta-estradiol, but not with 17alpha-estradiol. In 7-day-old mice, administration of varying concentrations of dexamethasone for 72 h resulted in a dose-dependent increase in the number of apoptotic osteoblasts as demonstrated by in situ terminal deoxynucleotidyltransferase-mediated deoxy-UTP-biotin nick end labeling staining of calvaria. A maximum of 22+/-1% apoptotic osteoblasts on the bone surface was found with 1 mg/kg BW dexamethasone compared with 2+/-1% in vehicle-treated mice. Injection of varying concentrations of 17beta-estradiol (0.5-5 mg/kg BW), but not 17alpha-estradiol, with 1 mg/kg dexamethasone produced a dose-dependent decrease in the number of apoptotic osteoblasts to 5+/-1% with 5 mg/kg 17beta-estradiol. Thus, glucocorticoid-induced apoptosis of osteoblasts may be prevented at least in part by 17beta-estradiol.     

Myostatin gene deletion prevents glucocorticoid-induced muscle atrophy.

Glucocorticoids mediate muscle atrophy in many catabolic states. Myostatin expression, a negative regulator of muscle growth, is increased by glucocorticoids and myostatin overexpression is associated with lower muscle mass. This suggests that myostatin is required for the catabolic effects of glucocorticoids. We therefore investigated whether myostatin gene disruption could prevent muscle atrophy caused by glucocorticoids. Male myostatin knockout (KO) and wild-type mice were subjected to dexamethasone treatment (1 mg/kg.d for 10 d or 5 mg/kg.d for 4 d). In wild-type mice, daily administration of low-dose dexamethasone for 10 d resulted in muscle atrophy (tibialis anterior: -15%; gastrocnemius: -13%; P < 0.01) due to 15% decrease in the muscle fiber cross-sectional area (1621 +/- 31 vs. 1918 +/- 64 microm(2), P < 0.01). In KO mice, there was no reduction of muscle mass nor fiber cross-sectional area after dexamethasone treatment. Muscle atrophy after 4 d of high-dose dexamethasone was associated with increased mRNA of enzymes involved in proteolytic pathways (atrogin-1, muscle ring finger 1, and cathepsin L) and increased chymotrypsin-like proteasomal activity. In contrast, the mRNA of these enzymes and the proteasomal activity were not significantly affected by dexamethasone in KO mice. Muscle IGF-I mRNA was paradoxically decreased in KO mice (-35%, P < 0.05); this was associated with a potentially compensatory increase of IGF-II expression in both saline and dexamethasone-treated KO mice (2-fold, P < 0.01). In conclusion, our results show that myostatin deletion prevents muscle atrophy in glucocorticoid-treated mice, by blunting the glucocorticoid-induced enhanced proteolysis, and suggest an important role of myostatin in muscle atrophy caused by glucocorticoids.     

Prolactin suppresses glucocorticoid-induced thymocyte apoptosis in vivo

The hypothesis that prolactin (PRL) functions as an immunomodulator was based on studies showing lymphocyte PRL receptors, and its effects on growth, differentiation, and apoptosis in lymphoid cells. However, studies of PRL (PRL-/-) and PRL receptor knockout mice indicated that PRL was not required for immune system development or function under basal conditions. Because PRL maintains survival in glucocorticoid (GC)-treated Nb2-T lymphocytes in vitro, and PRL and GCs are elevated during stress, we investigated whether PRL protected T cells in vivo from GC-induced apoptosis. Adrenalectomized mice [PRL -/-, undetectable PRL; pituitary grafted PRL-/- (PRL-/-Graft), elevated PRL; and PRL+/-, normal PRL] were treated with dexamethasone (DEX) or PBS. Thymocytes and splenocytes were isolated and annexin V labeling of phosphatidylserine, DNA fragmentation, and caspase-3 activation were assessed as indices of apoptosis. Total thymocytes and CD4+ and CD8+ T cells obtained from DEX-treated PRL-/- mice exhibited significantly increased annexin V binding. In contrast, binding was not altered by DEX in PRL-/-Graft thymocytes. In addition, DEX induced classic DNA fragmentation in PRL-/- thymocytes. Elevated serum PRL reduced this effect. Thymocytes from DEX-treated PRL-/- mice exhibited increased caspase-3 activation, which was inhibited in cells from PRL-/-Graft mice. Finally, elevated expression of X-linked inhibitor of apoptosis, XIAP, was observed in thymi from DEX-treated PRL -/-Graft mice. This is the first demonstration that elevated PRL antagonizes apoptosis in thymocytes exposed to GCs in vivo. These observations suggest that, under conditions of increased GCs, such as during stress, elevated PRL functions physiologically to maintain survival and function of T-lymphocytes.     

Resistance to glucocorticoid-induced apoptosis in lymphoblastic leukemia

Glucocorticoid (GC) resistance is a phenomenon of major significance in a number of clinical situations, including the therapy of lymphoid malignancies. Resistance may concern all, or just selected, GC effects, it may be absolute or just reflect a state of reduced sensitivity and, clinically relevant, be reversible or irreversible. Numerous molecular mechanisms can be envisaged acting either 'upstream' in the GC-triggered signaling pathway, i.e. at the level of the GC receptor (GR), or 'downstream' at the level of the GC-regulated genes responsible for individual GC effects. In lymphoid malignancies, GCs have anti-leukemic effects through the induction of apoptosis and/or cell cycle arrest. In this condition evidence for only a small number of mechanisms for GC resistance has been provided, mostly at the level of the GR. Herein, we review reports and hypotheses regarding 'upstream' and 'downstream' mechanisms for GC resistance in lymphoblastic leukemia and present an in vitro GC resistance model that might allow identification of resistance mechanisms.     

Mechanisms of glucocorticoid-induced Leydig cell apoptosis

The high levels of corticosterone (CORT) that are typically achieved during stress induce apoptotic death of Leydig cells. The intracellular mechanisms by which CORT acts on Leydig cells to induce apoptosis are unknown, and the present study tested for mediation by Fas ligand (FasL), a member of the tumor necrosis factor ligand family, in association with caspase activation. In addition, another apoptotic pathway involving in the participation of mitochondria was studied by evaluation of mitochondrial membrane potential (DeltaPsi) loss and generation of reactive oxygen species (ROS), which are early apoptotic events in many cell types. Rat Leydig cells were isolated from adrenalectomized rats on day 90 postpartum at 3, 6, 12, 24 and 48 h after the start of CORT administration (at a dose of 5 mg total/100 g body weight per day intraperitoneally in two daily injections starting 3 days after surgery). Both FasL and Fas receptor protein levels, analyzed by Western blot and fluorescent immunohistochemistry, increased at 6 h after the start of CORT administration, peaking at 24 h and declining thereafter. Leydig cell caspase-3 activity was analyzed in vitro. Low molecular weight DNA fragments that are characteristic of apoptosis were evident in Leydig cells by 12 h of exposure to 100 nM CORT in vitro, and the abundance of the fragments was more pronounced at 24 h. In the presence of a specific caspase inhibitor, Ac-DEVD-CHO, Leydig cell apoptosis was suppressed, corroborating the hypothesis that caspase-3 is involved in CORT-mediated cell death. Western blotting analysis revealed that procaspase-3 was present only at low levels in untreated control Leydig cells, and increased by 6 h of CORT administration. By 12 h, however, procaspase-3 was significantly reduced, and the cleaved, active caspase-3 forms appeared and increased through 24 h. These results indicated that FasL/Fas and caspase were implicated in CORT-mediated Leydig cell apoptosis. Decreased DeltaPsi and increased ROS generation were also measurable in Leydig cells for up to 2 days following CORT administration in vitro. These data indicate that activation of the Fas system, cleavage of procaspase-3, loss of DeltaPsi and increased ROS generation are all implicated in the process of CORT-induced Leydig cell death.     

Risedronate prevents the loss of microarchitecture in glucocorticoid-induced osteoporosis in rats

Osteoporosis is a severe complication of glucocorticoid treatment. Bisphosphonates are a powerful therapeutic option to prevent osteoporotic fractures. The aims of this study were: a) to determine bone alterations induced by therapy with glucocorticoids (GC); b) to establish the efficacy of risedronate (Ris) in the prevention of these effects. We studied 40 female Sprague-Dawley rats randomly divided into 4 groups of treatment, administered 3 times a week sc: 1. Control: vehicle of methylprednisolone (GC) + vehicle of Ris; 2. Ris: Ris 5 mug/kg body weight vehicle of GC; 3. GC: GC 7 mg/kg + vehicle of Ris; 4. GC+Ris: GC 7 mg/kg, Ris 5 microg/kg. Animals were treated for 30 days and then were sacrificed. Densitometry was performed at baseline and at the end of the treatment. Right tibiae were removed for histomorphometric analyses. The GC group showed a 7% decrease in bone density vs controls (p<0.05), while the GC+Ris group was associated with a 3.5% increase in bone density vs controls (p<0.05). In the GC group, histomorphometric evaluations showed reduced bone volume (BV/TV) and thinning of trabeculae (Tb.Th) vs controls (BV/TV: 31+/-1 vs 35+/-1%, p<0.05; Tb.Th: 43+/-2 vs 50+/-3 microm, p<0.01; Ac.f: 1.8+/-0.2 vs 1.6+/-0.3 N/yr). The GC+Ris group had increased BV/TV and Tb.Th, and reduced Ac.f vs the GC group. Ris also maintained trabecular microarchitecture. At the histological level, glucocorticoid-induced osteoporosis was characterized by decreased bone volume, reduced osteoblastic activity, and deterioration of microarchitecture. Ris counteracted these effects both by prolonging osteoblast activity, and by maintaining bone microarchitecture.     

Folic acid prevents and partially reverses glucocorticoid-induced hypertension in the rat

BACKGROUND: To investigate the effect of folic acid on the increased pressure in rats treated with either adrenocorticotropic hormone (ACTH) or dexamethasone (Dex), and to further investigate the role of tetrahydrobiopterin (BH(4)) in any effect of folic acid by comparing the effect of BH(4) with that of folic acid in Dex hypertension. METHODS: Male Sprague-Dawley (SD) rats were treated with saline, subcutaneous ACTH (0.2 mg/kg/d) or Dex (10 microg/rat/d). Folic acid (0.04 g/L drinking) or BH(4) (10 mg/kg/d intraperitoneally) was started before (prevention) and during (reversal) glucocorticoid treatment. RESULTS: Saline, BH(4), vehicle for BH(4), or folic acid alone did not change systolic blood pressure (BP). Systolic BP was increased by ACTH and Dex. Folic acid, but not BH(4), prevented the development of hypertension caused by ACTH and Dex treatment. The ACTH and Dex hypertension were partially reversed by folic acid. The BH(4) increased plasma total biopterin concentrations. The Dex decreased plasma NOx concentrations but had no effect on plasma biopterin concentrations. The ACTH and Dex increased plasma F(2)-isoprostane concentrations and decreased serum homocysteine concentrations compared with control but had no effect on serum folate concentrations. Folic acid increased serum folate concentrations compared with control but had no effect on homocysteine concentrations. CONCLUSIONS: Folic acid prevented and partially reversed both ACTH and Dex hypertension in rats without modifying the increase in plasma F(2)-isoprostane concentrations. Given that BH(4) failed to prevent ACTH or Dex hypertension, folic acid is unlikely to be acting through increased BH(4) production. The precise mechanism for the BP-lowering effect of folic acid in this model of hypertension remains to be determined.     

Spontaneous and glucocorticoid-induced apoptosis in human mature T lymphocytes

Glucocorticoid (GC)-induced apoptosis is a well-recognized physiologic regulator of murine T-cell number and function. We have analyzed its mechanisms in human mature T cells, which have been thought to be insensitive until recently. Peripheral blood T cells showed sensitivity to GC-induced apoptosis soon after the proliferative response to a mitogenic stimulation, and were also sensitive to spontaneous (ie, growth factor deprivation-dependent) apoptosis. CD8+ T cells were more sensitive to both forms than CD4+ T cells. Acquisition of sensitivity to GC-induced apoptosis was not associated with any change in number or affinity of GC receptors. Both spontaneous and GC-induced apoptosis were increased by the macromolecular synthesis inhibitors, cycloheximide (CHX) and puromycin. A positive correlation between the degree of protein synthesis inhibition and the extent of apoptosis was observed. Interleukin-2 (IL-2) IL-4, and IL-10 protected (IL-2 > IL-10 > IL-4) T cells from both forms of apoptosis in a dose-dependent manner. Our data suggest that spontaneous and GC-induced apoptosis regulate the human mature T-cell repertoire by acting early after the immune response and differentially affecting T-cell subsets.     

Sirt7 increases stress resistance of cardiomyocytes and prevents apoptosis and inflammatory cardiomyopathy in mice

Sirt7 is a member of the mammalian sirtuin family consisting of 7 genes, Sirt1 to Sirt7, which all share a homology to the founding family member, the yeast Sir2 gene. Most sirtuins are supposed to act as histone/protein deacetylases, which use oxidized NAD in a sirtuin-specific, 2-step deacetylation reaction. To begin to decipher the biological role of Sirt7, we inactivated the Sirt7 gene in mice. Sirt7-deficient animals undergo a reduction in mean and maximum lifespans and develop heart hypertrophy and inflammatory cardiomyopathy. Sirt7 mutant hearts are also characterized by an extensive fibrosis, which leads to a 3-fold increase in collagen III accumulation. We found that Sirt7 interacts with p53 and efficiently deacetylates p53 in vitro, which corresponds to hyperacetylation of p53 in vivo and an increased rate of apoptosis in the myocardium of mutant mice. Sirt7-deficient primary cardiomyocytes show a approximately 200% increase in basal apoptosis and a significantly diminished resistance to oxidative and genotoxic stress suggesting a critical role of Sirt7 in the regulation of stress responses and cell death in the heart. We propose that enhanced activation of p53 by lack of Sirt7-mediated deacetylation contributes to the heart phenotype of Sirt7 mutant mice.     

Rosiglitazone impacts negatively on bone by promoting osteoblast/osteocyte apoptosis

Thiazolidinediones (TZDs) increase peripheral tissue insulin sensitivity in patients with type 2 diabetes mellitus by activating the nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma). In bone marrow stromal cell cultures and in vivo, activation of PPARgamma by high doses (20 mg/kg/day) of TZDs has been reported to alter stem cell differentiation by promoting commitment of progenitor cells to the adipocytic lineage while inhibiting osteoblastogenesis. Here, we have examined the in vivo effects of low-dose rosiglitazone (3 mg/kg/day) on bone, administered to mice by gavage for 90 days. Rosiglitazone-treated mice had increased weight when compared with controls, with no significant alterations in serum levels of glucose, calcium or parathyroid hormone (PTH). Bone mineral density (BMD) at the lumbar vertebrae (L1-L4), ilium/sacrum, and total body was diminished by rosiglitazone treatment. Histologically, bone was characterized by decreased trabecular bone volume and increased marrow space with no significant change in bone marrow adipocity. Decreased osteoblast number and activity due to increased apoptotic death of osteoblasts and osteocytes was apparent while osteoclast parameters and serum levels of osteocalcin, alkaline phosphatase activity, and leptin were unaltered by rosiglitazone treatment. Therefore, the imbalance in bone remodeling that follows rosiglitazone administration arises from increased apoptotic death of osteogenic cells and diminished bone formation leading to the observed decrease in trabecular bone volume and BMD. These novel in vivo effects of TZDs on bone are of clinical relevance as patients with type 2 diabetes mellitus and other insulin resistant states treated with these agents may potentially be at increased risk of osteoporosis.     

Osteoprotegerin protects against generalized bone loss in tumor necrosis factor-transgenic mice

OBJECTIVE: To investigate the role of tumor necrosis factor (TNF) in systemic bone loss of chronic inflammatory conditions, such as rheumatoid arthritis (RA), and to address the therapeutic potential of osteoclast blockade. METHODS: We investigated systemic bone changes in human TNF transgenic (hTNFtg) mice, which spontaneously developed severe inflammatory arthritis. RESULTS: Osteodensitometry revealed a significant decrease in trabecular bone mineral density (BMD) (-37%) in hTNFtg mice, and histomorphometry revealed a dramatic loss of bone volume (-85%) compared with wild-type controls. Osteoclast-covered bone surface and serum levels of deoxypyridinoline crosslinks were significantly elevated, suggesting increased osteoclast-mediated bone resorption in hTNFtg mice. Osteoprotegerin (OPG) completely blocked TNF-mediated bone loss by increasing BMD (+89%) and bone volume (+647%). Most strikingly, formation of primary spongiosa was dramatically increased (+563%) in hTNFtg mice after OPG treatment. Osteoclast-covered bone surface and serum levels of deoxypyridinoline crosslinks were significantly decreased by OPG, suggesting effective blockade of osteoclast-mediated bone resorption. OPG did not influence levels of hTNF, TNF receptor I (TNFRI), interleukin-1beta (IL-1beta), and IL-6. However, OPG decreased bone formation parameters (osteoblast-covered bone surface and serum osteocalcin levels), which were elevated in hTNFtg mice. In contrast to OPG, bisphosphonates and anti-TNF treatment did not affect generalized bone loss in hTNFtg mice. Anti-TNF, however, did not affect levels of TNF and TNFRI at the concentrations tested. These data indicate that generalized bone loss due to increased TNF can be blocked by OPG. CONCLUSION: OPG may represent a potent tool for preventing generalized loss of bone mass in chronic inflammatory disorders, especially RA.     

C-peptide prevents hippocampal apoptosis in type 1 diabetes

To explore mechanisms underlying central nervous system (CNS) complications in diabetes, we examined hippocampal neuronal apoptosis and loss, and the effect of C-peptide replacement in type 1 diabetic BB/W rats. Apoptosis was demonstrated after 8 months of diabetes, by DNA fragmentation, increased number of apoptotic cells, and an elevated ratio of Bax/Bcl-xL, accompanied by reduced neuronal density in the hippocampus. No apoptotic activity was detected and neuronal density was unchanged in 2-month diabetic hippocampus, whereas insulin-like growth factor (IGF) activities were impaired. In type 1 diabetic BB/W rats replaced with C-peptide, no TdT-mediated dUTP nick-end labeling (TUNEL)-positive cells were shown and DNA laddering was not evident in hippocampus at either 2 or 8 months. C-peptide administration prevented the preceding perturbation of IGF expression and reduced the elevated ratio of Bax/Bcl-xL. Our data suggest that type 1 diabetes causes a duration-dependent programmed cell death of the hippocampus, which is partially prevented by C-peptide.     

Involvement of Bax protein in the prevention of glucocorticoid-induced thymocytes apoptosis by melatonin

The antiapoptotic effect of melatonin has been described in several systems. In this study, the antagonistic effect of the methoxyindole on dexamethasone-induced apoptosis in mouse thymocytes was examined. Melatonin decreased both DNA fragmentation, and the number of annexin V-positive cells incubated in the presence of dexamethasone. Analysis of the expression of the members of the Bcl-2 family indicated that the synthetic glucocorticoid increased Bax protein levels without affecting the levels of Bcl-2, Bcl-XL, Bcl-XS, or Bak. This effect correlated with an increase in thymocytes bax mRNA levels. Dexamethasone also increased the release of cytochrome C from mitochondria. All of these effects were reduced in the presence of melatonin, which was ineffective per se on these parameters. In addition, the involvement of cAMP on glucocorticoid/melatonin antagonism was examined. Both melatonin and dexamethasone decreased the levels of this nucleotide in mouse thymocytes, indicating that the antagonistic action between both hormones involves a cAMP-independent pathway. In summary, the present results suggest that the antiapoptotic effect of melatonin on glucocorticoid-treated thymocytes would be a consequence of an inhibition of the mitochondrial pathway, presumably through the regulation of Bax protein levels.     

Modification of glucocorticoid sensitivity by MAP kinase signaling pathways in glucocorticoid-induced T-cell apoptosis

OBJECTIVE: Glucocorticoid is widely used for the treatment of diseases such as hematological malignancies. Glucocorticoid sensitivity is different from person to person and the mechanism of the regulation of glucocorticoid sensitivity is not well known. Glucocorticoid resistance is a major clinical problem. METHODS AND RESULTS: Here, using glucocorticoid-induced T-cell apoptosis, a model system for the analysis of the mechanism of glucocorticoid action, we clarified that mitogen-activated protein kinases (MAPKs) modify glucocorticoid sensitivity, namely that the activation of extracellular signal-regulated protein kinase (ERK) and p38 MAP kinase reduce and enhance glucocorticoid sensitivity, respectively. CONCLUSION: These findings might provide new tools for overcoming glucocorticoid-resistance.     

Gefitinib prevents bleomycin-induced lung fibrosis in mice

RATIONALE: Transforming growth factor-alpha and epidermal growth factor (EGF), the ligands for EGF receptor (EGFR), stimulate fibroblast proliferation and play an important role in the pathogenesis of pulmonary fibrosis. Therefore, inhibition of the EGFR signal by an EGFR tyrosine kinase inhibitor (EGFR-TKI) may prevent pulmonary fibrosis. However, there is a possibility that blocking the EGFR signal may inhibit epithelial cell repair, thereby exaggerating lung fibrosis. OBJECTIVE: To investigate the effect of EGFR-TK inhibition on lung fibrosis. METHODS: We looked at the effects of the EGFR-TKIs gefitinib (20, 90, 200 mg/kg) and AG1478 (12 mg/kg) on a bleomycin-induced lung fibrosis model in mice. MEASUREMENTS AND MAIN RESULTS: Gefitinib prevented lung fibrosis at all three doses. Furthermore, in those mice that did not receive bleomycin treatment, gefitinib at 200 mg/kg did not induce lung fibrosis. Immunohistochemistry revealed that phosphorylation of EGFR in lung mesenchymal cells induced by bleomycin was inhibited by gefitinib. AG1478 also attenuated the lung fibrosis. In vitro studies further demonstrated that the addition of gefitinib or AG1478 suppressed the EGFR ligand-induced proliferation of lung fibroblasts. CONCLUSIONS: These findings suggest that, in the preclinical setting, EGFR-TKIs may have a protective effect on lung fibrosis induced by bleomycin. Because these molecular targeted drugs may have differing effects depending on species and individuals, a cautious interpretation is warranted.