The effect of a corticosteroid cream and a barrier-strengthening moisturizer in hand eczema. A double-blind, randomized, prospective, parallel group clinical trial.

BACKGROUND: Hand eczema is a common and persistent disease with a relapsing course. Clinical data suggest that once daily treatment with corticosteroids is just as effective as twice daily treatment. OBJECTIVES: The aim of this study was to compare once and twice daily applications of a strong corticosteroid cream in addition to maintenance therapy with a moisturizer in patients with a recent relapse of hand eczema. METHODS: The study was a parallel, double-blind, randomized, clinical trial on 44 patients. Twice daily application of a strong corticosteroid cream (betamethasone valerate 0.1%) was compared with once daily application, where a urea-containing moisturizer was substituted for the corticosteroid cream in the morning. The investigator scored the presence of eczema and the patients judged the health-related quality of life (HRQoL) using the Dermatology Life Quality Index (DLQI), which measures how much the patient's skin problem has affected his/her life over the past week. The patients also judged the severity of their eczema daily on a visual analogue scale. RESULTS: Both groups improved in terms of eczema and DLQI. However, the clinical scoring demonstrated that once daily application of corticosteroid was superior to twice daily application in diminishing eczema, especially in the group of patients with lower eczema scores at inclusion. CONCLUSIONS: Twice daily use of corticosteroids was not superior to once daily use in treating eczema. On the contrary, the clinical assessment showed a larger benefit from once daily treatment compared with twice daily, especially in the group of patients with a moderate eczema at inclusion.

Enhancement of bioavailability by lowering of fat content in topical formulations.

BACKGROUND: The cosmetic properties of topical formulations are important parameters for the adherence to treatment, where modern oil-in-water emulsions are considered more acceptable compared with ointments. After application of an emulsion to the skin, the concentration of active ingredients in the formulation residue on the skin will increase, due to evaporation of volatile ingredients. OBJECTIVES: The aim of the present study was to investigate the effect of changes in vehicle fatty content on the skin penetration of two active ingredients: benzyl nicotinate (BN) and betamethasone valerate (BV). METHODS: Formulations containing 0.5% BN and 0.3% BV in vehicles with different lipid content (10-80%) were applied in a randomized and double-blind manner to the forearm of healthy volunteers. The changes in skin colour (erythema and blanching) were then monitored visually and with a new noninvasive instrument. RESULTS: The BN formulation containing 10% fat induced erythema more rapidly and with higher intensity than the formulations with higher fat content. Increased efficacy was also observed from the low-fat content formulation of BV, which gave more blanching than the formulations with high fat content. CONCLUSIONS: The rate of penetration of the active ingredients was inversely related to the lipid content, i.e. simple changes of the cosmetic properties by modifications of the lipid content may affect the efficacy of a formulation.

Treatment with a barrier-strengthening moisturizing cream delays relapse of atopic dermatitis: a prospective and randomized controlled clinical trial.

BACKGROUND: Standard treatment of atopic dermatitis (AD) is based on topical glucocorticosteroids or calcineurin inhibitors to treat flares combined with moisturizer treatment to alleviate dry skin symptoms. Patients with AD have an abnormal skin barrier function, and strategies for reducing the risks for eczema would be to repair the barrier or prevent barrier dysfunction. OBJECTIVES: The objective of this study was to explore the time to relapse of eczema during a 26-week maintenance treatment with a urea containing moisturizer compared to no treatment (neither medical nor non-medicated preparations) after successful clearing of atopic lesions. The moisturizer has previously been shown to improve skin barrier function. METHODS: Patients applied betamethasone valerate (0.1%) on eczematous lesions during a 3-week period. Those with cleared eczema entered a 26-week maintenance phase, applying the moisturizer or left the previously affected area untreated. Upon eczema relapse, patients were instructed to contact the clinic and to have the relapse confirmed by the investigator. RESULTS: Fifty-five patients entered the study and 44 patients were included in the maintenance phase (22 using moisturizer twice daily and 22 using no treatment). Median time to relapse for patients treated with moisturizer was > 180 days (duration of the study) compared with 30 days for the no-treatment group. Sixty-eight per cent of the patients treated with the moisturizer and 32% of the untreated patients remained free from eczema during the observation period. CONCLUSIONS: Maintenance treatment with a barrier-improving urea moisturizer on previous eczematous areas reduced the risk of relapse to approximately one third of that of no treatment.

Sex differences in the effect of finasteride on acute ethanol withdrawal severity in C57BL/6J and DBA/2J mice.

The neurosteroid allopregnanolone (ALLO) is a potent positive modulator of GABAA receptors that can modulate ethanol (EtOH) withdrawal. The 5alpha-reductase inhibitor finasteride can block the formation of ALLO and other GABAergic neurosteroids and also reduce certain effects of EtOH. Treatment with finasteride during chronic EtOH exposure decreased EtOH withdrawal severity and blood EtOH concentrations (BECs), suggesting an additional effect of finasteride on EtOH pharmacokinetics. Thus, the purpose of the present study was to determine the effect of finasteride on acute EtOH withdrawal severity, to minimize the effect of finasteride on EtOH metabolism. Male and female C57BL/6J and DBA/2J mice received a pretreatment of finasteride (50 mg/kg i.p.) or vehicle 24 h prior to an injection of EtOH (4 g/kg i.p.) or saline. Handling-induced convulsions (HICs) were scored at baseline, and then over a 24 h period after EtOH or saline injection. In another experiment, plasma estradiol and corticosterone levels were assessed at selected time points (0, 2, 8, and 24 h). In a final study, retro-orbital blood samples were collected at 30, 60, 120, and 240 min post-EtOH administration to access finasteride's effects on EtOH clearance parameters. Pretreatment with finasteride increased acute EtOH withdrawal severity in female C57BL/6J and DBA/2J mice but decreased withdrawal severity in male mice of both strains. Finasteride did not alter BECs, EtOH clearance, estradiol, or corticosterone concentrations in a manner that appeared to contribute to the sex difference in finasteride's effect on acute EtOH withdrawal severity. These findings suggest that male and female C57BL/6J and DBA/2J mice differ in their sensitivity to changes in ALLO or other GABAergic neurosteroid levels during acute EtOH withdrawal. Sex differences in the modulation of GABAergic 5alpha-reduced steroids may be an important consideration in understanding and developing therapeutic interventions in alcoholics.

Anticonvulsive effects of kappa-opioid receptor modulation in an animal model of ethanol withdrawal.

Although the neurochemical mechanisms contributing to alcohol withdrawal seizures are poorly understood, withdrawal seizures probably reflect neuronal hyperexcitability resulting from adaptation to chronic alcohol. Altered kappa-Opioid receptor (KOP-R) signaling has been observed in multiple seizure types; however, a role for this system in ethanol withdrawal seizures has not been systematically characterized. We hypothesized that pharmacological manipulations of the KOP-R would alter withdrawal in mice selectively bred for differences in ethanol withdrawal severity. Withdrawal Seizure-Prone (WSP) and Withdrawal Seizure-Resistant (WSR) mice were made physically dependent using chronic ethanol vapor inhalation, and the effects of the KOP-R antagonist nor-binaltorphimine or agonist U-50,488H on withdrawal severity were examined. Pretreatment with nor-binaltorphimine significantly increased handling-induced convulsion (HIC) severity in withdrawing WSR mice, with no observable effects in withdrawing WSP mice. In contrast, U-50,488H significantly decreased HIC severity in WSP mice, with no effects in WSR mice. During extended withdrawal (i.e. hours 12+), a rebound hyperexcitability was observed in WSP mice given agonist. Thus, administration of a KOP-R antagonist increased withdrawal severity in mice normally resistant to withdrawal seizures, while a KOP-R agonist reduced convulsion severity in animals susceptible to withdrawal seizures. These observations are consistent with differences in the KOP-R system observed in these lines at the molecular level, and suggest the KOP-R system may be a promising therapeutic target for management of ethanol withdrawal seizures. Finally, these findings underscore the importance of determining the potential for rebound increases in withdrawal severity during later withdrawal episodes.

Serotonin transporter and receptor expression in osteocytic MLO-Y4 cells.

Neurotransmitter regulation of bone metabolism has been a subject of increasing interest and investigation. We reported previously that osteoblastic cells express a functional serotonin (5-HT) signal transduction system, with mechanisms for responding to and regulating uptake of 5-HT. The clonal murine osteocytic cell line, MLO-Y4, demonstrates expression of the serotonin transporter (5-HTT), and the 5-HT1A, and 5-HT2A receptors by real-time RT-PCR and immunoblot analysis. Immunohistochemistry using antibodies for the 5-HTT, and the 5-HT1A and 5-HT2A receptors reveals expression of all three proteins in both osteoblasts and osteocytes in rat tibia. 5-HTT binding sites were demonstrated in the MLO-Y4 cells with nanomolar affinity for the stable cocaine analog [125I]RTI-55. Imipramine and fluoxetine, antagonists with specificity for 5-HTT, show the highest potency to antagonize [125I]RTI-55 binding in the MLO-Y4 cells. GBR-12935, a relatively selective dopamine transporter antagonist, had a much lower potency, as did desipramine, a selective norepinephrine transporter antagonist. The maximal [3H]5-HT uptake rate in MLO-Y4 cells was 2.85 pmol/15 min/well, with a Km value of 290 nM. Imipramine and fluoxetine inhibited specific [3H]5-HT uptake with IC50 values in the nanomolar range. 5-HT rapidly stimulated PGE2 release from MLO-Y4 cells; the EC50 for 5-HT was 0.1 microM, with a 3-fold increase seen at 60 min. The rate-limiting enzyme for serotonin synthesis, tryptophan hydroxylase, is expressed in MLO-Y4 cells as well as osteoblastic MC3T3-E1 cells. Thus, osteocytes, as well as osteoblasts, are capable of 5-HT synthesis, and express functional receptor and transporter components of the 5-HT signal transduction system.

Mutations in signal sequence cleavage domain of preproparathyroid hormone alter protein translocation, signal sequence cleavage, and membrane-binding properties.

Signal sequences, known to mediate the targeting of nascent secreted proteins to membranes, share common structural domains: a positively charged amino-terminus, a hydrophobic core, and a signal cleavage domain. Mutations have been introduced into the cDNA encoding the signal sequence of the mammalian protein preproparathyroid hormone to analyze the roles played by the signal cleavage domain in secretion. Two mutant genes were constructed missing the entire six-residue propeptide sequence and several residues of the signal cleavage domain. The effects of these mutations on signal function were assessed after expression in clonal cell lines and in a transcription-linked translation system. Alterations in the signal cleavage domain resulted in reduced translocation and signal cleavage. Furthermore, in one mutant, the removal of the signal cleavage domain converted the signal into a membrane anchor sequence. The nonhydrophobic sequences at the end of the signal sequence thus crucially affect the translocation, cleavage, and membrane-binding properties of signal sequences.

Consequences of amino-terminal deletions of preproparathyroid hormone signal sequence.

PTH is initially synthesized as a larger precursor, containing a 25 amino acid signal sequence. Modification of cDNA encoding the hormone precursor resulted in the synthesis of proteins whose signal sequences were shortened at their amino termini. The effects of these mutations were analyzed using a cell-free translation system and rat pituitary GH4 cells in culture. Removal of the first six amino acids of the signal sequence had no effect on the efficiency or kinetics of protein processing as measured in the two assay systems. Mutants lacking 10 or 13 amino acids were not processed efficiently in the cells, nor were they translocated across microsomes in the cell-free translation system. These studies suggest that a modest change in the hydrophobic domain of the signal sequence, which might not have been predicted to alter function, led to a dramatic decline in signal activity.

Beta-adrenergic receptor kinase-like activity and beta-arrestin are expressed in osteoblastic cells.

Biologic responses to peptide calciotropic hormones, such as parathyroid hormone (PTH) and calcitonin, exhibit desensitization. As with most hormones, however, the mechanisms of desensitization are not completely understood. For the beta 2-adrenergic receptor (beta 2AR) system, which is coupled to adenylyl cyclase via the stimulatory guanine nucleotide-binding regulatory (G5) protein, homologous desensitization is mediated in part by a receptor-specific kinase (beta ARK) and a soluble cofactor (beta-arrestin). Recently, this system has been reported to be involved in rapid homologous desensitization of the PTH/parathyroid hormone receptor protein (PTHrP) receptor. We have identified the presence of this system in bone using reverse-transcriptase PCR. Nucleotide sequence of PCR fragments from ROS 17/2.8 cells revealed 100% identity with rat brain beta ARK1 and beta-arrestin 1 sequences. Northern analyses with RNA from ROS 17/2.8, UMR 106-H5 cells, and primary cultures of nontransformed neonatal rat calvariae demonstrated two mRNA species of 4 and 2.6 kilobases (kb) for beta ARK and 7.5 kb for beta-arrestin, comparable to those found in bovine brain. beta ARK-like activity was demonstrated in cytosolic extracts of the UMR 106-H5 cells by assessing phosphorylation of the retinal photoreceptor, rhodopsin, by the extracts. Phosphorylation was enhanced with light-activated rhodopsin and by bovine brain G beta gamma subunits; heparin inhibited phosphorylation. These findings are characteristic of beta ARK. Expression of beta-arrestin in the UMR 106-H5 cells was confirmed by immunoblot. Thus, osteoblastic cells express proteins, beta ARK, and beta-arrestin, which may regulate desensitization of calciotropic hormone receptors.

Interaction of nascent preproparathyroid hormone molecules with microsomal membranes.

To characterize the early steps in the interaction of nascent chains of preproparathyroid hormone (prepro-PTH) with the secretory apparatus, such truncated nascent chains still attached to ribosomes were tested for binding to microsomal membranes and cleavage by signal peptidase. Nascent chains of 114, 97, 88, 81, 70, and 59 residues were tested for their ability to bind tightly to membranes and to undergo signal sequence cleavage. Chains of 81 residues and longer bound tightly to the membranes and were cleaved by signal peptidase. The 88- and 81-residue precursors and their corresponding pro-proteins were less efficiently associated with the membranes than were the 114- and 97-residue precursors and their corresponding pro-proteins. The 70-residue chain bound to the membrane but was not cleaved. When this peptide was subsequently released from the ribosome with puromycin, it was cleaved by signal peptidase. The 59-residue chain bound only slightly to the microsomal membrane and was not cleaved by signal peptidase, even when the nascent peptide was released from the ribosome with puromycin. Thus the critical length for productive binding to microsomal membranes is between 59 and 70 residues; the length required for signal cleavage is between 70 and 81 residues.

Changes in insulin-like growth factor-binding protein expression and secretion during the proliferation, differentiation, and mineralization of primary cultures of rat osteoblasts.

Insulin-like growth factor-I (IGF-I) and IGF-II are secreted by the bone-forming osteoblast and have been shown to promote mitogenesis and/or differentiation of several of the cells involved in adult bone remodeling. The biological actions of the IGFs are modulated in a cell-specific manner by IGF-binding proteins (IGFBPs). All six IGFBPs are expressed by osteoblasts. Both in vitro and in vivo, osteoblasts progress through a developmental sequence from committed precursors to mature differentiated cells that form a mineralized extracellular matrix. We have examined IGFBP expression and secretion by rat calvarial cultures, a model system of osteoblast development, to correlate changes with the developmental stage. Differential expression and secretion of IGFBPs during osteoblast development were observed. Maximal IGFBP-2 and -5 messenger RNA (mRNA) expression occurred in proliferating preosteoblasts, whereas mature osteoblasts showed maximal expression of IGFBP-3, -4, and -6. Rat osteoblasts did not express IGFBP-1. Increases in IGFBP-2, -3, and -4 secretion lagged behind corresponding mRNA increases by 3-6 days. Whereas mRNA levels declined as the cultures mineralized, IGFBP secretion continued to increase. Inhibition of osteoblast proliferation, which promotes differentiation, resulted in an IGFBP secretory pattern that was consistent with that seen with mature cells. Conversely, an IGFBP secretion pattern characteristic of proliferating cells could be maintained for weeks if differentiation was inhibited. We conclude that the developmental stage of the osteoblast is an important determinant of IGFBP secretion. We propose that hormonal regulation that alters the developmental stage may secondarily affect IGFBP expression or secretion.

Regulation of osteoblastic gene expression by lead.

Although it is well recognized that lead accumulates in bone, skeletal tissue is considered primarily a sequestering compartment and not a site of toxic action for lead. However, exposure to lead is associated with impaired skeletal growth in children and reductions in indices of bone formation in laboratory animals. Osteoblastic ROS 17/2.8 cells were used in an effort to better understand the consequences of lead exposure on skeletal homeostasis. Studies on confluent cultures of ROS 17/2.8 cells revealed that lead (2-200 microM) had no effect on cell number or DNA and protein synthesis. However, alkaline phosphatase activity was reduced by lead in a dose- and time-dependent manner. Reductions in steady state alkaline phosphatase mRNA levels paralleled the lead-induced inhibition of enzyme activity. Moreover, lead exposure resulted in similar dose-dependent reductions in steady state type 1 procollagen and bone Gla protein mRNA levels. The effect of lead on osteoblastic gene expression in ROS 17/2.8 cultures, however, was selective in nature, as similar lead exposures resulted in no alterations in beta-actin or glyceraldehyde-3-phosphate dehydrogenase mRNA levels. These data demonstrate that lead, in the absence of over toxicity, specifically restricts the expression of certain aspects of the differentiated osteoblast phenotype. Such alterations in osteoblast function may contribute to the skeletal abnormalities observed in settings of lead intoxication.

Homologous androgen receptor up-regulation in osteoblastic cells may be associated with enhanced functional androgen responsiveness.

Although androgens have myriad effects on the skeleton, the regulation of androgen action in bone is not well understood. Androgen receptors (ARs) are known to play an important role in mediating androgen action. We have examined the effects of androgens and other sex steroids on AR levels in osteoblastic cells in vitro using two clonal human cell lines, SaOS-2 and U-2 OS. AR protein levels were quantitated both by specific androgen binding studies and Western analyses, and AR messenger RNA was measured with RNase protection assays. Potential changes in AR functionality was assessed by reporter assays. Treatment of osteoblastic cells with the nonaromatizable androgen 5alpha-dihydrotestosterone (DHT) increased specific androgen binding 2-to 4-fold. Similar increases in AR protein levels were documented by Western analysis in both cell lines. The androgen-mediated increase in receptor levels was time and dose dependent as well as androgen specific. Steady-state AR messenger RNA levels were also increased by DHT. When AR concentrations in osteoblastic cells were elevated with exogenous receptor, there was an enhancement of DHT responsiveness, measured by increased trans-activation of an androgen-responsive promoter. Thus, androgen exposure increased androgen receptor protein levels and specific androgen binding in osteoblastic cells. Androgen action as measured by androgen-mediated transcriptional activation is enhanced in the presence of elevated AR levels. Consequently, these studies have revealed an additional means by which androgens may modulate skeletal metabolism.

Transcriptional up-regulation of the human androgen receptor by androgen in bone cells.

Androgen regulation of androgen receptor (AR) expression has been observed in a variety of tissues, generally as inhibition, and is thought to attenuate cellular responses to androgen. AR is expressed in osteoblasts, the bone-forming cell, suggesting direct actions of androgens on bone. Here we characterized the effect of androgen exposure on AR gene expression in human osteoblastic SaOS-2 and U-2 OS cells. Treatment of osteoblastic cells with the nonaromatizable androgen 5alpha-dihydrotestosterone increased AR steady state messenger RNA levels in a time- and dose-dependent fashion. Reporter assays with 2.3 kilobases of the proximal 5'-flanking region of the human AR promoter linked to the chloramphenicol acetyltransferase gene in transfected cultures showed that up-regulation of AR promoter activity by androgen was time and dose dependent. Treatment with other steroid hormones, including progesterone, 17beta-estradiol, and dexamethasone, was without effect. The antiandrogen hydroxyflutamide completely antagonized androgen up-regulation. Thus, in contrast to many other androgen target tissues, androgen exposure increases steady state AR messenger RNA levels in osteoblasts. This regulation occurs at least partially at the level of transcription, is mediated by the 5'-promoter region of the AR gene, and is dependent on functional AR. These results suggest that physiological concentrations of androgens have significant effects on AR expression in skeletal tissue.

Functional cloning vectors for use in directional cDNA cloning using cohesive ends produced with T4 DNA polymerase.

This paper describes the construction of 'Prime' cloning vectors, which include phage lambda and plasmid vectors useful for functional cloning in oocytes, yeast, and mammalian cells, and their use in a 'Prime' cloning system. The system takes advantage of the very active and precise 3' exonuclease activity of T4 DNA polymerase to produce single-stranded (ss) ends (cut-back) of vector and insert DNA. This results in the highly efficient directional cloning of cDNA and PCR-amplified DNA. The system obviates the need to digest insert DNA with a restriction endonuclease to unveil cloning sites, and thus eliminates the chance of internal digestion of the insert DNA. The cloning of PCR-amplified DNA, which is sometimes difficult, is made routine with this system. The 'Prime' sequence is included in vector cloning sites and cDNA and PCR primers. The 'Prime' sequence was chosen so that the ss sticky ends are nonpalindromic and will hybridize only to the appropriate partners. This makes cloning with the 'Prime' system very efficient, because neither the vector nor insert DNA is lost to unproductive self-hybridization.

Reduced G-protein-coupled-receptor kinase 2 activity results in impairment of osteoblast function.

Rapid phosphorylation of many G-protein-coupled receptors (GPCRs) by G-protein-coupled receptor kinases (GRKs) accompanies stimulus-driven desensitization. Recent evidence suggests that GRKs and their associated arresting proteins, beta-arrestins, function as essential elements in the GPCR-mediated mitogen-activated protein (MAP) kinase signaling cascade. We investigated the interaction between GRKs and MAP kinase activation by growth factors in UMR 106-H5 osteoblastic cells stably expressing a dominant negative mutant of GRK2 (K220R). Expression of K220R in osteoblastic cells results in reduced cellular proliferation, both basally and in response to insulin-like growth factor-1 (IGF-1), and blunting of IGF-1- and EGF-induced MAP kinase activation. Reduced MAP kinase activation is not associated with alterations in IGF-1-receptor autophosphorylation. Both a constitutively active Ras mutant and PMA fully activate MAP kinase in K220R cells. We found that disruption of the GRK2 gene results in: (1) reduced osteoblast proliferation in response to growth factors, and (2) impaired receptor tyrosine kinase activation of mitogenic signaling pathways. Thus, GRK2 may regulate growth factor responsiveness in osteoblasts by modulating multiprotein complex formation following receptor tyrosine kinase activation.

Neurotransmitter action in osteoblasts: expression of a functional system for serotonin receptor activation and reuptake.

Neurotransmitter regulation of bone metabolism has been the subject of increasing interest and investigation. Because serotonin (5-HT) plays a role as a regulator of craniofacial morphogenesis, we investigated the expression and function of 5-HT receptors and the 5-HT transporter (5-HTT) in bone. Primary cultures of rat osteoblasts (rOB) and a variety of clonal osteoblastic cell lines, including ROS 17/2.8, UMR 106-H5, and Py1a, showed mRNA expression for 5-HTT as well as the 5-HT(1A), 5-HT(1D), 5-HT(2A), and 5-HT(2B) receptors by reverse transcription-polymerase chain reaction (RT-PCR) analysis. Protein expression of the 5-HT(1A), 5-HT(2A), and 5-HT(2B) receptors was confirmed by immunoblot. 5-HTT binding sites were assessed in ROS 17/2.8 and UMR 106-H5 cells by binding of the stable cocaine analog [125I]RTI-55, which showed a relatively high density of nanomolar affinity binding sites. Imipramine and fluoxetine, antagonists with specificity for 5-HTT, showed the highest potency to antagonize [125I]RTI-55 binding in ROS and UMR cells. GBR-12935, a relatively selective dopamine transporter antagonist, had a much lower potency, as did desipramine, a selective norepinephrine transporter antagonist. The maximal [3H]5-HT uptake rate in ROS cells was 110 pmol/10 min per well, with a K(m) value of 1.13 micromol/L. Imipramine and fluoxetine inhibited specific [3H]5-HT uptake with IC(50) values in the nanomolar range. In normal differentiating rOB cultures, 5-HTT functional activity was observed initially at day 25, and activity increased almost eightfold by day 31. In mature rOB cultures, the estimated density of [125I]RTI-55 binding sites was 600 fmol/mg protein. Functional downregulation of transporter activity was assessed after PMA treatment, which caused a significant 40% reduction in the maximal uptake rate of [3H]5-HT, an effect that was prevented by pretreatment with staurosporine. The affinity of 5-HT for the transporter was significantly increased following PMA treatment. We assessed the functional significance of expression of the 5-HT receptors by investigating the interaction between 5-HT and parathyroid hormone (PTH) signaling. 5-HT potentiates the PTH-induced increase in AP-1 activity in UMR cells. These results demonstrate that osteoblastic cells express a functional serotonin system, with mechanisms for responding to and regulating uptake of 5-HT.

Androgen inhibition of MAP kinase pathway and Elk-1 activation in proliferating osteoblasts.

Non-aromatizable androgens have significant beneficial effects on skeletal homeostasis independently of conversion to estradiol, but the effects of androgens on bone cell metabolism and cell proliferation are still poorly understood. Using an osteoblastic model with enhanced androgen responsiveness, MC3T3-E1 cells stably transfected with androgen receptor (AR) under the control of the type I collagen promoter (colAR-MC3T3), the effects of androgens on mitogenic signaling were characterized. Cultures were treated with the non-aromatizable androgen 5alpha-dihydrotestosterone (DHT) and the effects on osteoblast viability were determined as measured by an MTT assay. A complex response was observed in that continuous short-term DHT treatment enhanced osteoblast viability, but with longer-term DHT treatment inhibition was observed. The inhibition by DHT was prevented by the specific AR antagonist hydroxyflutamide, and was also observed in primary cultures of normal rat calvarial osteoblasts. In order to identify potential mediators of this effect, mitogenic pathway-specific cDNA microarrays were interrogated. Reduced hybridization of several genes important in MAP kinase-mediated signaling was observed, with the most dramatic effect on Elk-1 expression. Analysis of phosphorylation cascades demonstrated that DHT treatment inhibited phosphoERK1/2 levels, MAP kinase activation of Elk-1, Elk-1 protein and phosphoElk-1 levels, and downstream AP-1/luciferase reporter activity. Together, these data provide the first evidence that androgen inhibition of the MAP kinase signaling pathway is a potential mediator of osteoblast growth, and are consistent with the hypothesis that the MAP cascade may be a specific downstream target of DHT.

Osteoblast differentiation influences androgen and estrogen receptor-alpha and -beta expression.

Significant levels of estrogen and androgens circulate in men and women, and both play an important role in bone metabolism. While it is well established that either estrogen or androgen replacement therapy is effective at ameliorating bone loss associated with hypogonadism, recent evidence nevertheless suggests that estrogen and androgens have distinct molecular actions on the skeleton. In this study, we have employed normal rat calvarial osteoblast cultures to characterize relative expression profiles of estrogen (ERalpha and ERbeta) and androgen receptors (AR) during osteoblast differentiation. Normal osteoblast cultures can proceed through in vitro differentiation with distinct stages of proliferation, matrix maturation and mineralization in the appropriate differentiation medium containing ascorbic acid. Expression profiles of AR, ERalpha and ERbeta in primary cultures during osteoblast differentiation were characterized both by semi-quantitative relative RT-PCR and by Western analysis. In cultures induced to differentiate by growth in the presence of ascorbic acid, the expression profile for each receptor was unique during the course of differentiation. ERalpha levels were elevated during matrix maturation and then declined during mineralization. ERbeta expression was relatively constant throughout differentiation, exhibiting more constitutive expression. In contrast, AR levels were lowest during proliferation, and then increased throughout differentiation with highest levels in the most mature mineralizing cultures. Since steroid hormone action is generally mediated by specific cognate receptors, these results suggest that androgen actions may target cells during the mineralization stage of osteoblast differentiation, while estrogen action through either receptor isoform is more likely to affect osteoblasts earlier during matrix maturation. Interestingly, sex steroid receptor expression profiles did not exhibit the same patterns of regulation if osteoblast cultures were grown without ascorbic acid in medium that did not support extracellular matrix deposition. Thus, sex steroids may distinctly influence skeletal health by differential modulation of function during osteoblast differentiation.

Changes in IGF-I and -II expression and secretion during the proliferation and differentiation of normal rat osteoblasts.

IGF-I and -II have potent effects on proliferation and differentiation of osteoblasts in vitro. These cells secrete both IGFs and expression of these peptides is regulated by several of the hormones and growth factors that promote bone resorption and/or formation. However, the physiological role(s) of IGFs in the remodelling process of adult bone is still unclear. Some confusion may arise from results influenced, in part, by differences in the state of osteoblast development of in vitro cultures. Several laboratories have demonstrated that murine osteoblast cultures progress from proliferating preosteoblasts, to mature differentiated osteoblasts that form an extracellular matrix, to cultures that form a mineralized matrix. We have recently documented changes in IGF-binding protein expression and secretion in these cultures. To complement and extend this work, we have examined IGF-I expression and secretion and IGF-II expression during in vitro osteoblast development. Steady-state mRNA levels of both IGF-I and -II increased from the earliest time examined, day 5 in culture, to a maximum at day 11 and, thereafter, declined. IGF-I secreted into the medium also changed in a biphasic manner, but IGF-II could not be quantitated due to the sensitivity of our assay. Secretion of IGF-I was lowest between days 8 and 14. IGF-I secretion on day 5 was significantly greater than day 8. Similarly, IGF-1 secretion from day 17 to 26 was also greater than observed for days 8 to 14. If differentiation of the cells was inhibited, this late rise in IGF-I secretion was abolished.(ABSTRACT TRUNCATED AT 250 WORDS)