Antiestrogens specifically up-regulate bone morphogenetic protein-4 promoter activity in human osteoblastic cells.

Bone morphogenetic protein-4 (BMP-4) plays an important role in the onset of endochondral bone formation in humans, and a reduction in BMP-4 expression has been associated with a variety of bone diseases. Here we describe, by transient transfection assays in bone cells, that the human BMP-4 promoter recently characterized in our laboratory can be stimulated specifically by antiestrogens but not by estrogens or other steroid hormones. This activity is dependent on the presence of the estrogen receptor (ER)-alpha, although the promoter lacks a consensus estrogen-responsive element. No activity was observed in the presence of ERbeta, but synergy was observed when both ER subtypes were cotransfected. The observed stimulation of BMP-4 promoter activity by antiestrogens appeared bone cell specific and was reversed upon addition of estrogens. Since antiestrogens are known to be effective in hormone replacement therapies for postmenopausal women, this observation may help to develop new strategies for treatment and prevention of osteoporosis.

Homeobox proteins as signal transduction intermediates in regulation of NCAM expression by recombinant human bone morphogenetic protein-2 in osteoblast-like cells.

The role of homeobox genes in signaling of recombinant human bone morphogenetic protein-2 (rhBMP-2) was studied in osteoblast-like cells. Expression of several homeobox genes was decreased by rhBMP-2. The finding that this regulation of homeobox gene expression by rhBMP-2 was not dependent on protein synthesis suggests that homeobox proteins can act as direct intermediates in signal transduction of BMPs. Therefore, we studied the regulation of neural cell adhesion molecule (NCAM), which has previously been described as a target gene of both rhBMP-2 and homeobox proteins. We now show that in osteoblast-like cells, rhBMP-2 inhibits NCAM expression, while HOXC6 increases its expression, both acting via the same region of the promoter. As overexpression of HOXC6 could abolish effects of rhBMP-2 on NCAM promoter activity, these data show for the first time that members of the homeobox gene family may form direct functional intermediates in the signaling mechanism of the TGF-beta superfamily.

Cell-type-specific modulation of Hox gene expression by members of the TGF-beta superfamily: a comparison between human osteosarcoma and neuroblastoma cell lines.

Homeobox gene expression in osteoblast-like cells was investigated using the polymerase chain reaction (PCR). A total of 13 homeobox genes was detected in U-2 OS (human osteosarcoma) and MC3T3-E1 (mouse osteoblast) cells by sequencing cloned PCR products. Using specific primers, a different pattern of Hox gene expression was shown for the neuroblastoma cell line SK-N-SH relative to U-2 OS and MC3T3-E1. Additionally, we showed that expression of HOXC6 in U-2 OS and SK-N-SH was differentially regulated by rhBMP-2, TGF-beta and activin-A. This suggests that specific Hox genes may be target genes for TGF-beta superfamily members, and allows us to further understand the complex functions of these growth factors and how they relate to growth and development.

Genomic organization of the human bone morphogenetic protein-4 gene: molecular basis for multiple transcripts.

The structure of the human bone morphogenetic protein-4 (BMP-4) gene has been characterized from a genomic cosmid clone of about 38 kb. The transcriptional unit of the human BMP-4 gene is encoded by 5 exons and spans approximately 7 kb. The exon-intron organization of the human BMP-4 gene is similar to that of the mouse gene, with notable sequence differences in the 5' non-coding exons. The human BMP-4 gene has at least two functional promoters, which are used in a cell type specific manner. This observation is of fundamental relevance for understanding the specific role of BMP-4 in skeletal development and bone remodeling.

Neuron-glia interactions in the release of oxytocin and vasopressin from the rat neural lobe: the role of opioids, other neuropeptides and their receptors.

The release of the neurohormones oxytocin and vasopressin from the neural lobe into the circulation is regulated in a complex manner, which has only been partly elucidated. At the level of the neural lobe, regulation of release can occur by various endogenous compounds that act on specific receptors present on the nerve terminals themselves. In addition, release may be modulated by an alternative pathway in which the local glia cells, the pituicytes, are involved. It is especially the latter pathway that is discussed in detail in this commentary.

Technical aspects of opioid receptor localization: detection of opioid receptor proteins by immunocytochemistry or with a biotinylated dynorphin analog.

Opioid receptors were localized at the cellular level, using either anti-opioid receptor antibodies or a biotinylated opioid ligand. In addition, a simple method was developed for selection of second antisera on their potencies to detect particular monoclonal antibodies (mAbs). Most anti-opioid receptor antibodies tested were not able to recognize the opioid receptor in frozen or fixed tissue sections, which was in contrast with their ability to recognize opioid receptors in isolated membrane fractions. However, one batch of anti-idiotypic mAbs gave a good immunocytochemical staining. Distribution of immunoreactivity suggested that these antibodies recognized more than one opioid receptor subtype. After very short fixation times, staining with a biotinylated kappa-selective ligand (DAKLIB) could be observed in the neural and intermediate lobe of pituitary. This binding could be displaced by non-biotinylated DAKLI. The pattern of DAKLIB staining in the neural lobe had the appearance of binding to both nerve fibres and astrocytes. The present results show successful staining of tissue sections with anti-idiotypic antibodies and with a biotinylated ligand. The specificity is discussed in the light of control experiments, pharmacological data and previous studies.

Characterization of opioid binding sites in the neural and intermediate lobe of the rat pituitary gland by quantitative receptor autoradiography.

Previous studies have suggested an involvement of enkephalins in regulation of oxytocin (OXT) and vasopressin (AVP) release, which seems to disagree with the very low affinities of Met- and Leu-enkephalin for the kappa opioid receptor. As opioid receptors in the neural lobe exclusively exist of kappa receptors, we studied the binding characteristics of larger pro-enkephalin derived peptides for opioid binding sites in the neural lobe by means of light microscopic receptor autoradiography. In addition, the pharmacological characteristics of opioid binding sites in the neural lobe were compared with those in other parts of the pituitary. In the neural as well as the intermediate lobe both high and low affinity 3H-bremazocine binding sites were present. Binding to these sites was completely displaceable by both naloxone and nor-binaltorphimine suggesting that these sites represent kappa opioid receptors. Also with regard to selectivity and affinity characteristics to other ligands, opioid binding sites in the neural and intermediate lobe were quite similar. In the anterior lobe a very low level of bremazocine binding was present, which could not be displaced by nor-binaltorphimine. Displacement studies with pro-enkephalin and pro-dynorphin derived peptides showed that both groups of peptides could bind to opioid binding sites in the neural and intermediate lobe. Especially the relatively large pro-dynorphin and pro-enkephalin derived peptides, such as dynorphin 1-17 and BAM22, appeared to be very potent ligands for these opioid binding sites and were much more potent than smaller fragments, such as dynorphin 1-8, and Met- and Leu-enkephalin. These results contradict the existence of a mismatch in the neural (and intermediate) lobe with regard to the local type of opioid peptides and receptors present.

Dynorphin 1-17 delays the vasopressin induced mobilization of intracellular calcium in cultured astrocytes from the rat neural lobe.

Opioid peptides are present in nerve terminals in the rat neural lobe where they partially coexist with vasopressin. Morphological findings suggest that these neuropeptides are released onto pituicytes, which is in agreement with a possible role for the pituicyte in oxytocin and vasopressin release from the neural lobe. Pituicytes in culture respond to vasopressin with a mobilization of calcium from intracellular stores. In the present study this vasopressin induced increase in intracellular free calcium levels was both delayed and decreased by pre-exposure to dynorphin 1-17, while dynorphin 1-17 by itself did not affect basal calcium levels. All effects of dynorphin 1-17 could be blocked with naloxone. The present results suggest that opioid receptors are present on pituicytes and are coupled to a second messenger pathway by which opioid peptides may inhibit inositol phosphate dependent calcium mobilization by other neuropeptides, such as vasopressin.

Immunocytochemical localization of neuropeptide FF (FMRF amide-like peptide) in the hypothalamo-neurohypophyseal system of Wistar and Brattleboro rats by light and electron microscopy.

Neuropeptide FF (F8Famide, FMRFamide-like, or morphine modulating peptide) immunoreactivity was localized by light and electron microscopy in the hypothalamo-neurohypophyseal system of Wistar and Brattleboro rats. In Wistar rats neuropeptide FF was present in part of the magnocellular neurones of the paraventricular and supraoptic nuclei in which it was coexpressed with vasopressin. Neuropeptide FF containing fibres were present in the paraventricular and the supraoptic nuclei, and in the central part of the neural lobe. At the electron microscopic level, neuropeptide FF containing nerve terminals in the neural lobe formed synaptoid contacts exclusively with pituicytes. No neuropeptide FF containing neurovascular contacts or contacts with other neuronal structures were observed. In contrast with Wistar rats, neuropeptide FF was almost completely absent in cell bodies of the paraventricular and supraoptic nuclei, and in fibres of the neural lobe in Brattleboro rats. Only a few solitary cells could be observed in these structures. The present results demonstrate that neuropeptide FF coexists with vasopressin within the hypothalamo-neurohypophyseal system. As we did not observe neuropeptide FF containing neurovascular contacts, neuropeptide FF containing nerve terminals probably have a local function within the neural lobe. Neuropeptide FF may be involved in the modulation of oxytocin and vasopressin release, with the pituicyte as an intermediate cell.

Immunoelectron microscopic demonstration of oxytocin and vasopressin in pituicytes and in nerve terminals forming synaptoid contacts with pituicytes in the rat neural lobe.

A pre-embedding immunoelectron microscopic technique was used to obtain morphological evidence for a role of oxytocin and vasopressin in the regulation of their own or each others release from the neural lobe. No synaptoid contacts of oxytocin- or vasopressin-containing axons with other neuronal structures were observed. However, synaptoid contacts of oxytocin- and vasopressin-containing nerve terminals and Herring bodies with pituicytes were frequently observed. These findings suggest that the pituicyte may participate in auto- and/or cross-regulation of oxytocin and vasopressin release. Moreover, oxytocin and vasopressin precursor-derived peptides were found in the cytoplasm of some pituicytes, an unexpected finding that will be discussed.

Isoproterenol-stimulated release of beta-endorphin and related peptides from the rat pituitary neurointermediate lobe in vitro: evidence for preferential release of certain molecular forms of beta-endorphin.

The intermediate lobe of the pituitary gland synthesizes the multifactorial precursor molecule pro-opiomelanocortin (POMC), from which, through a process of post-translational enzymatic processing, beta-endorphin-(1-31) (beta E) and a variety of N alpha-acetylated and C-terminally shortened forms of this peptide are generated. Using an in vitro superfusion system, the release of these endorphins from intact rat neurointermediate lobes (NILs) was investigated under basal and isoproterenol (ISO) stimulated conditions. Superfusion of NILs with the beta-adrenergic agonist ISO (30 min pulse) resulted in a rapid, sustained and concentration-dependent stimulation of the release of beta E-like immunoreactivity (beta E-IR) over basal as determined with an antiserum directed against the C-terminus of the beta E- (1-31) sequence (10(-6) M: + 145%; 10(-7) M: + 73%; 10(-8) m: + 41%). The release of N(alpha)-acetylated-endorphin-like immunoreactivity (AcE-IR) was stimulated to a similar extent. These effects of ISO were antagonized by the competitive alpha-adrenoceptor antagonist propranolol in a concentration-dependent manner, indicating the involvement of alpha-adrenoceptors. The beta-related peptides released from the NILs under basal and ISO-stimulated conditions were further characterized, based on their retention times in a reversed-phase HPLC system and their reactivity with specific antisera recognizing respectively the midportion of beta E, the N-terminus of acetylated endorphins, the C-terminus of tau-endorphin (beta E-(1-17); tau E), or the C-terminus of alpha-endorphin (beta E-(1-16); alpha E). In HPLC fractionated superfusates 10 peaks were resolved that reacted with the midportion beta E antiserum. In superfusates collected under basal conditions, three major peaks possessed chromatographical and immunological characteristics of Ac beta E-(1-26), Ac beta E- (1-27) Ac beta E-(1-31). In addition, a prominent peak was found eluting around the retention time of beta E-(1-31), that contained both acetylated and non-acetylated material. Six smaller peaks were observed, with the characteristics of beta E-(1-26) and beta E-(1-27) (these peptides were not resolved with the HPLC system used), Ac tau E, tau E, Aa alpha E, and des-tyrosine-alpha E (DT alpha E), respectively. In superfusates collected during superfusion of NILs with ISO (10(-6) M) all peaks were increased. However, those eluting as beta E-(1-31), beta E-(1-26)/beta E-(1-27), Ac beta E-(1-26) and Ac tau E appeared to be preferentially stimulated.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of chronic treatment with haloperidol and bromocriptine on the processing of beta-endorphin to gamma- and alpha-endorphin in discrete regions of the rat pituitary gland and brain.

beta-Endorphin is the putative precursor molecule of gamma- and alpha-endorphin. To investigate whether long-term changes in the activity of cells producing beta-endorphin are paralleled by alterations in the enzymatic processing of beta-endorphin, the effects of chronic treatment of rats with dopamine (DA) receptor ligands were examined on the content of immunoreactivity of beta-, gamma- and alpha-endorphin of dissected regions of the pituitary gland and the brain. Treatment with the DA receptor antagonist, haloperidol, resulted in a significant increase in the concentration of immunoreactivity for beta-, gamma-, and alpha-endorphin in the neurointermediate lobe, and of beta-endorphin in the anterior lobe of the pituitary gland. Levels of immunoreactivity of alpha-melanotropin and beta-endorphin in plasma were elevated, but those of corticosterone were decreased. This indicates that, in the intermediate lobe, both the biosynthetic and the secretory activity of cells producing beta-endorphin had increased, whereas in the anterior lobe, the secretory activity of beta-endorphin cells had decreased. No effects were observed on the ratios beta-endorphin/gamma-endorphin and beta-endorphin/alpha-endorphin in the intermediate lobe. In the anterior lobe however, the ratio beta-endorphin/alpha-endorphin had significantly increased. The effects of chronic treatment with the DA receptor agonist, bromocriptine, on levels of hormones in pituitary and plasma were opposite to those induced by haloperidol. In the brain, treatment with haloperidol selectively increased the content of immunoreactivity for beta-, gamma- and alpha-endorphin of the hypothalamus and the hippocampus and did not affect levels of peptides in the other regions of the brain studied.(ABSTRACT TRUNCATED AT 250 WORDS)