Interleukin 17A: a Janus-faced regulator of osteoporosis.

Interleukin (IL)-17A is a well-described mediator of bone resorption in inflammatory diseases, and postmenopausal osteoporosis is associated with increased serum levels of IL-17A. Ovariectomy (OVX) can be used as a model to study bone loss induced by estrogen deficiency and the role of IL-17A in osteoporosis development has previously been investigated using various methods to inhibit IL-17A signaling in this model. However, the studies show opposing results. While some publications reported IL-17A as a mediator of OVX-induced osteoporosis, others found a bone-protective role for IL-17 receptor signaling. In this study, we provide an explanation for the discrepancies in previous literature and show for the first time that loss of IL-17A has differential effects on OVX-induced osteoporosis; with IL-17A being important for cortical but not trabecular bone loss. Interestingly, the decrease in trabecular bone after OVX in IL-17A knock-out mice, was accompanied by increased adipogenesis depicted by elevated leptin levels. Additionally, the bone marrow adipose tissue expanded, and the bone-turnover decreased in ovariectomized mice lacking IL-17A compared to ovariectomized WT mice. Our results increase the understanding of how IL-17A signaling influences bone remodeling in the different bone compartments, which is of importance for the development of new treatments of post-menopausal osteoporosis.

Increased bone mass in a mouse model with low fat mass.

Mice with impaired acute inflammatory responses within adipose tissue display reduced diet-induced fat mass gain associated with glucose intolerance and systemic inflammation. Therefore, acute adipose tissue inflammation is needed for a healthy expansion of adipose tissue. Because inflammatory disorders are associated with bone loss, we hypothesized that impaired acute adipose tissue inflammation leading to increased systemic inflammation results in a lower bone mass. To test this hypothesis, we used mice overexpressing an adenoviral protein complex, the receptor internalization and degradation (RID) complex that inhibits proinflammatory signaling, under the control of the aP2 promotor (RID tg mice), resulting in suppressed inflammatory signaling in adipocytes. As expected, RID tg mice had lower high-fat diet-induced weight and fat mass gain and higher systemic inflammation than littermate wild-type control mice. Contrary to our hypothesis, RID tg mice had increased bone mass in long bones and vertebrae, affecting trabecular and cortical parameters, as well as improved humeral biomechanical properties. We did not find any differences in bone formation or resorption parameters as determined by histology or enzyme immunoassay. However, bone marrow adiposity, often negatively associated with bone mass, was decreased in male RID tg mice as determined by histological analysis of tibia. In conclusion, mice with reduced fat mass due to impaired adipose tissue inflammation have increased bone mass.

ERα expression in T lymphocytes is dispensable for estrogenic effects in bone.

Estrogen treatment has positive effects on the skeleton, and we have shown that estrogen receptor alpha (ERα) expression in cells of hematopoietic origin contributes to a normal estrogen treatment response in bone tissue. T lymphocytes are implicated in the estrogenic regulation of bone mass, but it is not known whether T lymphocytes are direct estrogen target cells. Therefore, the aim of this study was to determine the importance of ERα expression in T lymphocytes for the estrogenic regulation of the skeleton using female mice lacking ERα expression specifically in T lymphocytes (Lck-ERα-/-) and ERαflox/flox littermate (control) mice. Deletion of ERα expression in T lymphocytes did not affect bone mineral density (BMD) in sham-operated Lck-ERα-/- compared to control mice, and ovariectomy (ovx) resulted in a similar decrease in BMD in control and Lck-ERα-/- mice compared to sham-operated mice. Furthermore, estrogen treatment of ovx Lck-ERα-/- led to an increased BMD that was indistinguishable from the increase seen after estrogen treatment of ovx control mice. Detailed analysis of both the appendicular (femur) and axial (vertebrae) skeleton showed that both trabecular and cortical bone parameters responded to a similar extent regardless of the presence of ERα in T lymphocytes. In conclusion, ERα expression in T lymphocytes is dispensable for normal estrogenic regulation of bone mass in female mice.

Roles of transactivating functions 1 and 2 of estrogen receptor-alpha in bone.

The bone-sparing effect of estrogen is primarily mediated via estrogen receptor-α (ERα), which stimulates target gene transcription through two activation functions (AFs), AF-1 in the N-terminal and AF-2 in the ligand binding domain. To evaluate the role of ERα AF-1 and ERα AF-2 for the effects of estrogen in bone in vivo, we analyzed mouse models lacking the entire ERα protein (ERα(-/-)), ERα AF-1 (ERαAF-1(0)), or ERα AF-2 (ERαAF-2(0)). Estradiol (E2) treatment increased the amount of both trabecular and cortical bone in ovariectomized (OVX) WT mice. Neither the trabecular nor the cortical bone responded to E2 treatment in OVX ERα(-/-) or OVX ERαAF-2(0) mice. OVX ERαAF-1(0) mice displayed a normal E2 response in cortical bone but no E2 response in trabecular bone. Although E2 treatment increased the uterine and liver weights and reduced the thymus weight in OVX WT mice, no effect was seen on these parameters in OVX ERα(-/-) or OVX ERαAF-2(0) mice. The effect of E2 in OVX ERαAF-1(0) mice was tissue-dependent, with no or weak E2 response on thymus and uterine weights but a normal response on liver weight. In conclusion, ERα AF-2 is required for the estrogenic effects on all parameters evaluated, whereas the role of ERα AF-1 is tissue-specific, with a crucial role in trabecular bone and uterus but not cortical bone. Selective ER modulators stimulating ERα with minimal activation of ERα AF-1 could retain beneficial actions in cortical bone, constituting 80% of the skeleton, while minimizing effects on reproductive organs.

Estrogen inhibits GH signaling by suppressing GH-induced JAK2 phosphorylation, an effect mediated by SOCS-2.

Oral estrogen administration attenuates the metabolic action of growth hormone (GH) in humans. To investigate the mechanism involved, we studied the effects of estrogen on GH signaling through Janus kinase (JAK)2 and the signal transducers and activators of transcription (STATs) in HEK293 cells stably expressing the GH receptor (293GHR), HuH7 (hepatoma) and T-47D (breast cancer) cells. 293GHR cells were transiently transfected with an estrogen receptor-alpha expression plasmid and luciferase reporters with binding elements for STAT3 and STAT5 or the beta-casein promoter. GH stimulated the reporter activities by four- to sixfold. Cotreatment with 17beta-estradiol (E(2)) resulted in a dose-dependent reduction in the response of all three reporters to GH to a maximum of 49-66% of control at 100 nM (P < 0.05). No reduction was seen when E(2) was added 1-2 h after GH treatment. Similar inhibitory effects were observed in HuH7 and T-47D cells. E(2) suppressed GH-induced JAK2 phosphorylation, an effect attenuated by actinomycin D, suggesting a requirement for gene expression. Next, we investigated the role of the suppressors of cytokine signaling (SOCS) in E(2) inhibition. E(2) increased the mRNA abundance of SOCS-2 but not SOCS-1 and SOCS-3 in HEK293 cells. The inhibitory effect of E(2) was absent in cells lacking SOCS-2 but not in those lacking SOCS-1 and SOCS-3. In conclusion, estrogen inhibits GH signaling, an action mediated by SOCS-2. This paper provides evidence for regulatory interaction between a sex steroid and the GHJAKSTAT pathway, in which SOCS-2 plays a central mechanistic role.

A transgenic model to determine the physiological role of liver-derived insulin-like growth factor I.

Insulin-like growth factor-I (IGF-I) has important growth promoting and metabolic effects and is expressed in virtually every tissue of the body. The highest expression is found in the liver but the physiological role of liver-derived IGF-I is unknown. It has been difficult to separate the endocrine effects of liver-derived IGF-I from the autocrine/paracrine effects of locally produced IGF-I in peripheral tissues. Therefore, we have developed a mouse model with a liver-specific inducible deletion of the IGF-I gene. The liver-IGF-I deficient mouse have dramatically reduced (>80%) serum IGF-I levels, demonstrating that the major part of serum IGF-I is liver-derived. Surprisingly, liver-IGF-I deficient mice demonstrate a normal appendicular skeletal growth up to at least 12 months of age despite the dramatic decrease in circulating IGF-I levels, indicating that liver-derived IGF-I is not required for appendicular skeletal growth. However, the adult axial skeletal growth is clearly reduced in the liver-IGF-I deficient mice. Furthermore, the amount of cortical bone is reduced due to decreased radial growth of the cortical bone while the amount of trabecular bone is unchanged in the liver-IGF-I deficient mice. The decreased levels of circulating IGF-I are associated with increased serum levels of growth hormone (GH), indicating a role for liver-derived IGF-I in the negative feedback regulation of GH secretion. Measurements of factors regulating GH-secretion in the pituitary and in the hypothalamus revealed an increased expression of growth hormone releasing hormone (GHRH) and growth hormone secretagogue (GHS) receptors in the pituitary of liver-IGF-I deficient mice. This in turn results in an increased sensitivity to systemically administered GHRH and GHS, demonstrating that the regulatory action of liver-derived IGF-I on GH secretion is at the pituitary rather than at the hypothalamic level. The liver is an important metabolic organ and liver-IGF-I deficient mice are markedly hyperinsulinemic and yet normoglycemic, consistent with an adequately compensated insulin resistance. Interestingly, liver-IGF-I deficient mice display a reduced age-dependent fat mass accumulation compared with control mice. In conclusion, liver-derived IGF-I is important for carbohydrate- and lipid-metabolism and for the regulation of GH-secretion at the pituitary level. Furthermore, it regulates adult axial skeletal growth and cortical radial growth while it is not required for appendicular skeletal growth.

Autoantibodies against the angiotensin receptor (AT1) in patients with hypertension.

Sera from patients with malignant essential hypertension (n = 14), malignant secondary hypertension mainly attributable to renovascular diseases (n = 12) and renovascular diseases without malignant hypertension (n = 11) and from normotensive healthy blood donors (n = 35) were studied for the presence of autoantibodies against G-protein-coupled cardiovascular receptors. Autoantibodies against the angiotensin II receptor (AT1) were detected in 14, 33, 18 and 14% of patients with malignant essential hypertension, malignant secondary hypertension, renovascular diseases and control patients, respectively. Sensitivity of the enzyme immunoassay was assessed as 5 microg/ml IgG. Patients did not show antibodies against bradykinin (B2) or angiotensin II subtype 2 (AT2) receptors. Autoantibodies affinity-purified from positive patients localized AT receptors in Chinese hamster ovary transfected cells, and displayed a positive chronotropic effect on cultured neonatal rat cardiomyocytes. These results demonstrate the existence of autoantibodies against a functional extracellular domain of human AT1 receptors in patients with malignant hypertension, and suggest that these autoantibodies might be involved in the pathogenesis of malignant hypertension.

Osteoprotegerin mRNA is increased by interleukin-1 alpha in the human osteosarcoma cell line MG-63 and in human osteoblast-like cells.

Osteoprotegerin (OPG) is a soluble receptor for the Osteoprotegerin-Ligand (OPGL) which is expressed on osteoblasts and mediates the signal for osteoclast differentiation. In the present study we demonstrate that OPG mRNA levels in MG-63 cells are increased in a dose-dependent manner after 8 h of treatment with IL-1 alpha (338 +/- 53% over control at 25 U/ml). Interleukin-6 (IL-6), under similar culture conditions, does not affect OPG mRNA levels. Time-course studies show that IL-1 alpha (25 U/ml) causes a transient increase of OPG mRNA levels in MG-63 cells, peaking after 4 h of treatment. An increase of the OPG transcript occurs in hOB cells at 0.5 h which is still present after 24 h of IL-1 alpha treatment. In MG-63 cells neither basal-nor IL-1 alpha-induced OPG mRNA levels are altered by the translational inhibitor cycloheximide. These results suggest that expression of OPG in osteoblasts may be regulated by IL-1 alpha.

Small animal imaging with pinhole single-photon emission computed tomography.

BACKGROUND: High resolution spatial details of the distribution of activity in three dimensions is required to evaluate the localization and dosimetric properties of radiolabelled monoclonal antibodies in tumors and normal tissues. Planar imaging of small animals with a resolution of 5-10 mm is usually the imaging modality of choice. The authors investigated high resolution single-photon emission computed tomographic (SPECT) imaging, based on a rotating pinhole scintillation camera. Although the sensitivity of the pinhole collimator is low, several radionuclides offer suitable decay properties to perform pinhole SPECT, especially in conjunction with high activity levels used in radioimmunotherapy. METHODS: Transverse, sagittal, and coronal sections were reconstructed using a three-dimensional cone-beam algorithm, which is a generalization of the two-dimensional fan-beam filtered backprojection algorithm. Before reconstruction, the pinhole projections were corrected for the decay of the radionuclide, geometric and intrinsic efficiency variations of the camera system, and center of rotation shift. RESULTS: The spatial resolution at 50 mm from the pinhole collimator with 3.3 mm aperture was 3.4 mm, and the sensitivity 7.2 c/s microCi for technetium-99m. With the 2 mm collimator the resolution was 2.2 mm, and the sensitivity was 2.6 c/s/microCi. To show the spatial resolution in vivo, a rat was injected with 185 MBq of technetium-99m-methylene diphosphonate or with 5 mCi technetium-99m-hexamethylpropylene amine oxime. The bone structures were well delineated in the methylene diphosphonate image, and in the hexamethylpropylene amine oxime image, the brain was nicely shown. For comparison a magnetic resonance image for the same section was done. CONCLUSIONS: High resolution SPECT imaging with the pinhole collimator provides mapping of the activity in three-dimensions, needed for more detailed biodistribution data and to perform more accurate dosimetry.

Effect of metoprolol on activity of beta-adrenoceptor coupled to guanine nucleotide binding regulatory proteins in adriamycin-induced cardiotoxicity.

Prevention of cardiotoxicity without interfering with the therapeutic efficacy of adriamycin is a very crucial question. We have investigated the activity of beta-adrenoceptor coupled to guanine nucleotide binding regulatory proteins (G-proteins) and Ca(2+)-ATPase activity in experimental adriamycin-induced cardiotoxicity and the influence of metoprolol treatment on these variables. Adriamycin was administered to rats intravenously as a single dose of 6 mg/kg, and metoprol was continuously given by means of implanted osmotic pumps. beta-Adrenoceptor characteristics were measured by radioligand-binding experiments and by basal and stimulated adenylyl cyclase activity. Northern blot and dot blot analysis was used to quantify G-protein mRNA. It was shown that adriamycin did not induce any change in the total beta-adrenoceptor density, nor did the high affinity agonist binding to beta-adrenoceptor change. Adriamycin did not induce any alteration in the amount of mRNA encoding for stimulatory (Gs) or inhibitory (Gi) G-proteins. Also, basal and stimulated adenylyl cyclase activities were identical in the different experimental groups. In contrast, the Ca(2+)-ATPase was shown to increase in adriamycin-treated rats compared to control rats (45 +/- 3.8 versus 23 +/- 1.2 mumol Pi/mg/h, P less than .01). Metoprolol was shown to normalize this increase (29 +/- 2.1 mumol Pi/mg/h). Thus, it may be concluded that in experimental adriamycin-induced cardiotoxicity, despite Ca(2+)-overloading, the beta-adrenoceptor-G protein-adenylyl cyclase system remains intact. Metoprolol seems to prevent Ca(2+)-overloading independently of the beta-adrenoceptors studied here.

The effect of diabetes on phosphatidylinositol turnover and calcium influx in myocardium.

Diabetes was induced in rats by administration of streptozotocin. Diabetes occurred within 24 h after treatment. Two forms of diabetes were studied, an acute form (4 days) and a chronic form (2 months). In a separate experiment the effect of insulin and an aldose reductase inhibitor on acute diabetes was studied. Phosphoinositide labelling was done in biopsies of heart with [3H] myo-inositol. It was shown that the incorporation of myo-inositol amounted to about 65% in acute diabetes and 80% in chronic diabetes compared to age-matched controls. The incorporation both in atria and ventricles was affected in a similar way. Muscarinic receptor-mediated phosphatidylinositol breakdown and release of myo-Ins-1 P (myo-inositol 1-phosphate) was unaffected in diabetic hearts in the chronic model. In hearts of diabetic ketotic animals uncoupling of the muscarinic receptor from the phosphoinositide metabolism was apparent. Calcium net influx was significantly reduced in both acute and chronic diabetes compared to age-matched controls. Insulin supplementation to acute diabetic animals significantly improved phosphoinositide labelling with [3H] myo-inositol. No improvement was seen in calcium transport. An aldose reductase inhibitor also facilitated phosphoinositide labelling without improving calcium transport. It is suggested that phosphoinositide metabolism and calcium entry through the slow inward current are independent of one another and the former is sensitive to insulin. It is suggested that insulin by regulating the pool of phosphoinositides and release of endogenous calcium may modulate cardiac function.

Sexual function in prostatic cancer patients treated with radiotherapy, orchiectomy or oestrogens.

Sexual function in prostatic carcinoma patients was studied in 12 patients from each of three treatment groups: radiotherapy, orchiectomy and oestrogen treatment. Significant deterioration occurred in all groups. Although erectile potency was preserved in 9 of 12 patients treated with radiotherapy, 7 of these had a marked reduction in the frequency of sexual activity. Men subjected to orchiectomy or oestrogen treatment were seldom capable of having intercourse or of experiencing orgasm. However, oestrogen-treated men continued sexual activity with their partner more often than orchiectomised subjects. Patients receiving oestrogen treatment scored significantly higher for mental depression than those in the other two treatment groups.