Localization of insulin-like growth factor-I mRNA in rat brain by in situ hybridization--relationship to IGF-I receptors.

Recent evidence has demonstrated regional synthesis of insulin-like growth factor I (IGF-I) in rat brain, which is also known to contain widespread specific type I IGF receptors. In order to precisely define sites of IGF-I mRNA synthesis, and their relationship to IGF-I receptor sites, we have applied the techniques of in situ hybridization and in vitro receptor autoradiography in rat brain. Frozen sections of adult rat brain and liver were hybridized with 32P-labeled cDNA inserts for human IGF-I (780 base pairs) or a positive control transthyretin cDNA (1430 base pairs) probe, or a series of negative probes, followed by film or emulsion autoradiography. Receptor autoradiography was performed on similar sections using 125I-IGF-I in buffer, some chambers containing excess unlabeled IGF-I. Hybridization of IGF-I probe was clearly seen only in three major brain regions: the olfactory bulb, hippocampus and cerebellum, whereas transthyretin only hybridized to choroid plexus as expected, and other probes showed no hybridization. In olfactory bulb, hybridization was greatest in the internal granular and mitral cell layers, with lower levels in the glomerular layer, where IGF-I receptors were concentrated. In hippocampus, hybridization was to pyramidal cells of Ammon's horn in CA1 and CA2 layers and dentate gyrus, with some labeling in CA3. IGF-I receptors were most dense in CA2, CA3, CA4, and dentate gyrus. In cerebellum, hybridization was to the granule cell layer, with IGF-I receptors primarily in the adjacent molecular layer. We have clearly demonstrated precise sites of local IGF-I synthesis in adult rat brain, adjacent to, and sometimes overlapping sites of high density IGF-I receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin receptor expression in bone.

Insulin is known to be an important osteotropic hormone. To date, no study has specifically addressed the possibility that insulin receptor expression may be regulated by differentiation in bone. We report a heterogeneous distribution of insulin receptor (IR) within neonatal rat calvaria using a specific monoclonal antibody to the beta-subunit of the rat insulin receptor (CT-1). Specific binding of CT-1 to mature osteoblasts was demonstrated, with little binding over periosteal tissues or osteocytes. Using enzymatically derived subpopulations of calvarial cells, we showed a correlation between alkaline phosphatase activity and insulin-stimulated 2-deoxyglucose (2-DOG) uptake and increased 125I-insulin binding. Since primary calvarial cultures contain many cell types, we compared 125I-insulin binding, insulin-stimulated 2-DOG uptake, and Northern blot analysis of IR mRNA in the clonal preosteoblast-like cell line UMR 201-10B and the mature osteoblast cell line UMR 106-01. It is shown that UMR 106-01 cells possess higher levels of IR mRNA, insulin binding, and insulin-stimulated glucose uptake, and that insulin up-regulated expression of mRNA of the glucose transporter GLUT1 by 3-fold. In contrast, insulin binding was negligible in UMR 201-10B cells, which expressed much lower levels of IR mRNA. UMR 201-10B cells did not possess an insulin-sensitive glucose uptake system, although they express GLUT1 mRNA. These data are consistent with the hypothesis that, as in muscle and fat, insulin receptor expression correlates with the stage of osteoblast differentiation in vivo and in vitro.

A novel orthotopic model of breast cancer metastasis to bone.

Breast cancer affects approximately one woman in twelve and kills more women than any other cancer. If detected early, patients have a five year survival rate of 66%, but once metastatic disease has developed, there is no effective treatment. About 70% of patients with metastatic disease have bone involvement, while lungs and liver are the other common targets. Bone metastases cause severe pain, pathological fractures and hypercalcaemia and thus are a significant clinical problem. The development of new therapies for metastatic breast carcinoma depends on a better understanding of the mechanism of homing of the tumour cells to bone, liver and lungs and the factors required for their growth in these organs. Research on mechanisms of breast cancer metastasis, particularly to bone, has relied on in vitro studies or on tumour models in which the inoculation route is designed to promote delivery of tumour cells to a specific organ. Metastases in bone are achieved by inoculation into the right ventricle of the heart. To our knowledge there has been no report of a model of metastatic spread from the mammary gland to distant sites which reliably includes bone. In this paper, we describe our recent development of a novel murine model of metastatic breast carcinoma. The new model is unique in that the pattern of metastatic spread closely resembles that observed in human breast cancer. In particular, these murine breast tumours metastasise to bone from the primary breast site and cause hypercalcaemia, characteristics not normally found in murine tumours, but common in human disease. Furthermore, in a preliminary characterisation of this model, we show that secretion of parathyroid hormone-related protein, a role for which has been implicated in breast cancer spread to bone, correlates with metastasis to bone. This model therefore provides an excellent experimental system in which to investigate the factors that control metastatic spread of breast cancer to specific sites, particularly bone. The special advantage of this system is that it involves the whole metastasis process, beginning from the primary site. Existing models consider mechanisms that pertain to growth of tumour once the site has been reached. An understanding of the regulation of these factors by potential therapeutic agents could lead to improvement in therapies designed to combat metastatic disease. For the first time, this development will allow exploration of the molecular basis of site-specific metastasis of breast cancer to bone in a clinically relevant model.

Temporal expression of PTHrP during endochondral bone formation in mouse and intramembranous bone formation in an in vivo rabbit model.

Expression of parathyroid hormone-related protein (PTHrP) messenger RNA (mRNA) and protein was investigated throughout the developmental progression of endochondral bone formation in mouse and intramembranous bone formation in an in vivo model in rabbit, using in situ hybridization and immunohistochemistry. Endochondral bone formation was investigated in a developing embryo, newborn, and adult mouse. In fetal long bones through to newborn (day 7), PTHrP mRNA and protein were consistently expressed in chondrocytes within the proliferative, transitional, and hypertrophic zones. In addition, high levels of PTHrP were also detected in osteoblasts on the surface of trabecular bone surfaces. Similarly, at the adult stage (week 7), PTHrP mRNA and protein were consistently expressed in chondrocytes at epiphyseal ends of the subarticular cartilage, within cortical periosteum, as well as in osteoblasts located at the metaphyseal trabecular bone surfaces. Using an in vivo intramembranous bone formation model in rabbits, expression of PTHrP mRNA and protein was demonstrated in preosteoblasts prior to trabecular bone formation (1-week bone harvest). As bone formed (2-, 3-, and 4-week bone tissue harvests), PTHrP mRNA and protein were highly expressed in actively synthesizing osteoblasts and in those osteocytes embedded within the superficial layers of the bone matrix. Lining osteoblasts and osteocytes buried deeply in the bone matrix displayed weak or no signal for PTHrP. The pattern of spatial and temporal expression of PTHrP demonstrated in cartilage cells and osteoblasts in the two systems suggests an important role of PTHrP in both endochondral and intramembranous bone formation.

Insulin receptor expression in primary and cultured osteoclast-like cells.

Skeletal growth is the net product of coordinated bone formation and resorption. Insulin is known to stimulate bone formation by actions on osteoblasts. It is not known whether insulin receptors are present on osteoclasts, or whether insulin regulates osteoclastic function. We present here immunocytochemical evidence of insulin receptor expression by mature mono- and multinucleated murine osteoclast-like cells generated in vitro, and in primary neonatal rat and mouse osteoclasts. Radiolabeled studies indicated that progressive enrichment of osteoclast-like cells in coculture was associated with increased insulin binding. When osteoclast-like cells generated in vitro were plated onto dentine slices, insulin dose-dependently inhibited pit formation by up to 80%, suggesting a role for insulin in osteoclast function. These data are consistent with an effect of insulin on bone resorption in addition to those previously recognized on bone formation, actions that together result in net bone growth.

Localization of RANKL (receptor activator of NF kappa B ligand) mRNA and protein in skeletal and extraskeletal tissues.

RANKL (receptor activator of NFkappaB ligand) is a membrane-associated osteoblastic molecule, and along with macrophage-colony-stimulating factor, is crucial for osteoclast formation. RANKL is known to be strongly expressed in osteoblasts and lymphoid tissues. We have sought to determine the skeletal and extraskeletal sites of production of RANKL mRNA and protein using the techniques of in situ hybridization and immunohistochemistry. Expression of RANKL mRNA and protein were determined in the developmental progression of endochondral bone formation in mouse, intramembranous bone formation in a rabbit model (mRNA only), in human giant cell tumors of bone, and at extraskeletal sites in the mouse. RANKL mRNA was expressed in prehypertrophic and hypertrophic chondrocytes at day E15 embryonic mouse long bone, and its expression was maintained at these sites throughout development. In newborn and adult mice, high levels of RANKL mRNA were expressed in mesenchymal cells of the periosteum and in mature osteoblasts, while megakaryocytes within the marrow microenvironment expressed RANKL mRNA from 1 week of age. Immunohistochemical analysis revealed a similar localization pattern of RANKL protein at the sites described. In the intramembranous bone formation model, RANKL mRNA was expressed in mesenchymal cells and in actively synthesizing osteoblasts, but not in flattened lining osteoblasts or late osteocytes. Expression of RANKL mRNA and protein in osteoclasts was variable with those within resorption lacunae showing the strongest signal/staining. Likewise, expression varied in osteoclasts from giant cell tumor of bone with a minority of tartrate-resistant acid phosphatase-positive multinucleated cells having no detectable RANKL mRNA or protein. In extraskeletal tissues, RANKL mRNA and protein were detected in the brain, heart, kidney, skeletal muscle, and skin throughout mouse development, suggesting the possibility of several other functions of the molecule. RANKL was also developmentally regulated, as evidenced by its expression in the intestine, liver, and lung at E15 and newborn mouse but not in the adult.

Fos production in retrogradely labelled neurons of the lamina terminalis following intravenous infusion of either hypertonic saline or angiotensin II.

The lamina terminalis consists of neurons which are activated by both osmotic and angiotensinergic stimuli and which project axons to many sites including regions of the hypothalamus responsible for vasopressin production. Combination of retrograde neuronal tracing procedures with the identification of Fos protein following discrete stimuli shows populations of neurons, projecting to the supraoptic nuclei, which are preferentially activated by intravenous infusion of either hypertonic saline or angiotensin II. Following infusion of hypertonic saline, the greatest percentage of neurons both labelled with cholera toxin-gold and having elevated levels of Fos protein occurred in that part of the lamina terminalis called the organum vasculosum lamina terminalis. Conversely, angiotensin infusion resulted in greatest numbers of Fos and cholera toxin-gold-labelled neurons in the subfornical organ with fewer double-labelled cells represented in the other components of the lamina terminalis, the median preoptic nucleus and the organum vasculosum lamina terminalis. While these data do not support more than a general separation of the functions examined among neurons of the lamina terminalis, they do highlight a discrete group of osmoresponsive neurons in the dorsal cap of the organum vasculosum lamina terminalis. These cells, by virtue of their response to infusions of hypertonic saline and their axonal connections to regions of the hypothalamus responsible for vasopressin production, are likely candidates for cerebral osmoreceptors.

Noradrenaline (gamma) and ATP responses of innervated and non-innervated rat cerebral arteries.

1. The distribution of sympathetic adrenergic nerves on the rat middle cerebral artery and on the arterioles which originated from it was determined by use of gloxylic histochemistry. 2. Whereas the middle cerebral artery and proximal arterioles arising from this artery received a sympathetic innervation, the distal regions of the same arterioles were devoid of innervation. 3. The arteries and arterioles which were innervated were depolarized by noradrenaline in the combined presence of alpha- and beta-adrenoceptor antagonists. Those which were not innervated were not depolarized by noradrenaline. 4. ATP depolarized all arteries and arterioles examined. 5. These observations are discussed with respect to the similarities and differences between gamma-adrenoceptors and P2 purinoceptors.

Efferent neural pathways of the lamina terminalis subserving osmoregulation.

Studies in rats and sheep show that neurons in the CVOs of the lamina terminalis provide extensive neural input to the vasopressin-containing cells of the supraoptic nucleus. This input is both by direct pathways and via a synapse in the MnPO which also has projections to the vasopressin-containing cells of the SON. Neurons throughout the lamina terminalis (including possible osmoreceptors in the OVLT and subfornical organ) are activated by systematic hypertonicity. It is likely that in response to hypertonicity they signal the SON and PVN to release vasopressin and elsewhere to elicit other osmoregulatory responses such as thirst and the excretion of sodium.

Neurons in the median preoptic nucleus of the rat with collateral branches to the subfornical organ and supraoptic nucleus.

A novel pathway between the subfornical organ and the supraoptic nucleus involving the collateral branches of cell bodies situated in the lamina terminalis has been studied. Fluorogold was injected into the supraoptic nucleus and rhodamine-labelled microspheres into the subfornical organ of rats. Nineteen % of neurons in the median preoptic nucleus and 30% of neurons in the OVLT projecting to the subfornical organ also had axons extending to the supraoptic nucleus. These pathways may represent a novel trajectory for the rely of information from the lamina terminalis to the supraoptic nucleus.

Projections from the subfornical organ to the supraoptic nucleus in the rat: ultrastructural identification of an interposed synapse in the median preoptic nucleus using a combination of neuronal tracers.

The subfornical organ, along with other regions of the lamina terminalis, may contain osmoreceptors and is likely to be a site of action of blood-borne angiotensin II. The neural pathways by which these stimuli lead to vasopressin secretion, have been suggested to extend from the subfornical organ to hypothalamic sites of vasopressin production either directly or via synapses in an intervening nucleus such as the median preoptic nucleus. In the present study, cholera toxin conjugated to horseradish peroxidase (CT/HRP) or colloidal gold (CT/Au) has been injected, respectively, into the subfornical organ and supraoptic nucleus of the same animal. The anterograde and retrograde transport of the toxin from these two sites has made possible the identification, at the ultrastructural level, of a synapse in the median preoptic nucleus interposed in the pathway between the subfornical organ and the supraoptic nucleus. Moreover, the presence of retrogradely transported CT/HRP and CT/Au in the same neurone in the median preoptic nucleus indicates that some neurones in this nucleus have axons with collateral branches to both the subfornical organ and supraoptic nucleus. Either or both of these pathways may transmit information related to the tonicity of the blood or circulating levels of angiotensin II to sites in the hypothalamus.

Identification of neural pathways activated in dehydrated rats by means of Fos-immunohistochemistry and neural tracing.

The distribution of Fos-immunoreactivity (Fos-IR) was studied in the brains of rats deprived of water for 24 or 48 h and compared with that in brains of water-replete rats. Intense Fos-IR was observed in many neurons of the median preoptic nucleus (MnPO), organum vasculosum of the lamina terminalis (OVLT), supraoptic nucleus and hypothalamic paraventricular nucleus. There was less intense and sparse Fos-IR in the subfornical organ. In water-replete rats, Fos-IR was absent or very low in these regions. In other rats, cholera toxin B-gold conjugate was microinjected bilaterally into the supraoptic nucleus to identify retrogradely labelled neurons in the lamina terminalis projecting to the supraoptic nucleus. Approximately 30% of these retrogradely labelled neurons in the OVLT and MnPO also exhibited Fos-IR after 48 h of water deprivation. These data show that neurons in the MnPO, OVLT and, to a lesser extent, the subfornical organ probably play an important role in homeostatic responses to dehydration, such as vasopressin secretion.

Identification of osmoresponsive neurons in the forebrain of the rat: a Fos study at the ultrastructural level.

The aims of this study are twofold. The first is to describe the ultrastructural morphology of putative osmoreceptors concentrated in the ventral aspect of the lamina terminalis in the rat forebrain. The second is to determine whether or not these neurons lie within an area which lacks a blood-brain barrier, i.e. the organum vasculosum lamina terminalis. The results describe a compact population of neurons in the ventral part of the lamina terminalis which both respond to an osmotic challenge and project directly to the supraoptic nucleus. Injection of horseradish peroxidase into the circulation, as a marker to define areas of the brain without a blood-brain barrier, indicates that these neurons are in the dorsal aspect of the organum vasculosum of the lamina terminalis. An ultrastructural analysis of the neurons in this area, which respond to an osmotic challenge with an elevation of Fos protein, show them to have no specific morphological characteristics which differentiate them from other, non-responsive neurons in the organum vasculosum of the lamina terminalis. However, one possible exception is that osmotically sensitive neurons have a less indented nucleus, suggesting that they are in a more active state than their non-osmotically sensitive neighbours. It is concluded that neurons in this region of the brain are candidate structures for the "receptors" which mediate vasopressin release in response to an osmotic challenge. The response of only a subset of neurons in the organum vasculosum of the lamina terminalis to an osmotic stimulus, despite an apparent morphological homogeneity and the ability of blood borne agents to reach all parts of the structure suggests that osmoresponsiveness is conferred by unique membrane properties or intracellular processing events. The presence of synaptic input to osmoresponsive cells indicates a potential for integration of other inputs at this level.

Distribution of angiotensin II receptor binding in the spinal cord of the sheep.

The distribution of angiotensin II binding sites has been mapped at segmental levels throughout the spinal cord of the sheep using in vitro autoradiographic methods. Binding of 125I-[Sar1.Ile8] Ang II is most prominent in the lateral horns of the thoracolumbar and sacral regions containing the sympathetic and parasympathetic preganglionic neurons respectively. Binding is also present in the dorsal horns of the grey matter, in the central canal region, dorsal root ganglia and associated with non-neuronal elements such as the ependyma surrounding the central canal, and blood vessels. Displacement with receptor antagonists specific for AT1 and AT2 subtypes, indicates that angiotensin II receptors in the spinal cord are of the AT1 type. These data help to interpret the physiological actions of angiotensin II in the spinal cord, particularly with respect to its autonomic components.

Subfornical organ lesion decreases sodium appetite in the sodium-depleted rat.

The effect of subfornical organ (SFO) lesion on various models of ingestive behaviour was investigated in rats. Intake of water after 24 h water deprivation or systemic administration of hypertonic NaCl were not altered by SFO lesions. Intake of food or water after 24 h of food deprivation were not altered by SFO lesions. Intake of NaCl after furosemide-induced Na depletion was decreased by ablation of the SFO. This decrease in Na intake was ameliorated by pretreatment with a low dose of captopril. These results suggest that the SFO is involved in Na intake after Na depletion, but not in water or food intake following periods of water or food deprivation, respectively. The observation that a low dose of captopril can eliminate the decrease in Na appetite which occurred subsequent to SFO lesion suggests that other brain areas may also participate in Na-depletion-induced Na appetite.

The distribution and ultrastructure of sensory elements in the baroreceptor region of the truncus arteriosus of the lizard Trachydosaurus rugosus.

The proximal truncus arteriosus of the lizard Trachydosaurus rugosus was studied with light-, fluorescence- and electron-microscopical techniques. Three vessels comprised the truncus; the pulmonary, left aortic, and carotico-aortic arteries. Right and left truncal nerves, each derived from the ipsilateral vagus nerve, innervated the truncus, particularly its proximal 3 mm. Ultrastructurally, the nerves had a variety of appearances: some were clearly adrenergic, c-type or p-type. A number of profiles contained large numbers of mitochondria and were classified as sensory. Some profiles defied exact classification, having characteristics common to two different types of profile. Within the outer medial layers, profiles up to 7 micrometers in diameter were found. These contained large numbers of mitochondria, myelin bodies and structures intermediate between the two. In addition, the profiles contained large amounts of glycogen and small numbers of vesicles. These nerve fibres were classified as baroreceptors, since they closely resemble carotid sinus and aortic arch baroreceptors in mammals. Large numbers of chromaffin cells were found, particularly in the common wall of the pulmonary and left aortic arteries. Many of these cells emitted a long tapering process, which sometimes entered a nearby nerve bundle. Sensory, p-type and c-type profiles, but not adrenergic profiles, made extensive close contacts with chromaffin cells.

Expression of osteoclast differentiation factor at sites of bone erosion in collagen-induced arthritis.

OBJECTIVE: To investigate the cellular mechanism of bone destruction in collagen-induced arthritis (CIA). METHODS: After induction of CIA in DA rats, a histologic study of the advanced arthritic lesion was carried out on whole, decalcified joints from the hindpaws of affected animals. To conclusively identify osteoclasts, joint tissue sections were stained for tartrate-resistant acid phosphatase (TRAP) enzyme activity, and calcitonin receptors (CTR) were identified using a specific rabbit polyclonal antibody. The expression of messenger RNA (mRNA) for the osteoclast differentiation factor (also known as receptor activator of nuclear factor kappaB ligand [RANKL]) was investigated using in situ hybridization with a specific riboprobe. RESULTS: TRAP-positive and CTR-positive multinucleated cells were invariably detected in arthritic lesions that were characterized by bone destruction. Osteoclasts were identified at the pannus-bone and pannus-subchondral bone junctions of arthritic joints, where they formed erosive pits in the bone. TRAP-positive multinucleated cells were detected within synovium and at the bone erosive front; however, CTR-positive multinucleated cells were present only at sites adjacent to bone. RANKL mRNA was highly expressed in the synovial cell infiltrate in arthritic joints, as well as by osteoclasts at sites of bone erosion. CONCLUSION: Focal bone erosion in CIA is attributed to cells expressing definitive features of osteoclasts, including CTR. The expression of RANKL by cells within inflamed synovium suggests a mechanism for osteoclast differentiation and activation at sites of bone erosion. Inhibitors of RANKL may represent a novel approach to treatment of bone loss in rheumatoid arthritis.

Expression of parathyroid hormone-related protein in cells of osteoblast lineage.

The expression of parathyroid hormone-related protein (PTHrP) was studied in a range of cell cultures representative of the osteoblast lineage and in rat calvarial sections. Primary newborn rat calvarial cells, a rat preosteoblastic cell line (UMR 201), a mouse stromal cell line (ST 2), a mouse calvaria-derived osteoblastic cell line (KS 4), and rat osteosarcoma cell lines (UMR 106-01 and -06), all expressed PTHrP when examined by reverse transcription polymerase chain reaction (RT-PCR). Using a radioimmunoassay we also demonstrated PTHrP in the conditioned medium of the cultured cells, with the exception of UMR 106-01 and -06 cells. Treatment of UMR 201 cells with all-trans-retinoic acid which induces them to acquire a more differentiated phenotype, also led to a time-dependent decrease in PTHrP mRNA levels as determined by RT-PCR, Northern blot analysis, and in situ hybridization. Decreased PTHrP levels in the conditioned medium of the treated cells was also observed. These results suggested that PTHrP production might be greater in less mature osteoblasts. Examination of the populations obtained from newborn rat calvariae by sequential collagenase digestion revealed that the early digests exhibited low ALP activity, low expression of PTH/PTHrP receptor mRNA, and no adenylate cyclase response to PTHrP(1-34). These populations showed the highest level of mRNA and production of PTHrP. Cells from later digests, the "osteoblast-rich" populations, had reduced PTHrP expression. Immunohistochemistry and in situ hybridization in sections of newborn rat calvariae showed PTHrP expression in cuboidal osteoblasts located adjacent to bone and in spindle-shaped cells in the periosteal region. It is concluded that PTHrP is produced by cells of the osteoblast lineage, supporting the hypothesis that PTHrP may function physiologically as a paracrine factor in bone.

Parathyroid-hormone-related protein in sarcoidosis.

Parathyroid-hormone-related protein (PTHrP) is the main mediator of the humoral hypercalcemia of malignancy. It is also detected in many normal adult and fetal tissues. Altered calcium metabolism occurs in sarcoidosis, and two cases of sarcoidosis with hypercalcemia and elevated plasma PTHrP are described. An archival study of 20 lymph node biopsies with the pathological diagnosis of sarcoidosis was performed. Immunohistochemistry using a polyclonal antiserum to human PTHrP and in situ hybridization using a riboprobe to human PTHrP were performed on the lymph node biopsies. Immunohistochemistry for PTHrP was also performed on the biopsies from the two cases with elevated plasma levels. Immunohistochemical analysis detected PTHrP in macrophages within granulomata in 17 of the 20 (85%) biopsies. In situ hybridization detected a positive signal for messenger RNA in the granulomata of 11 of 19 (58%) biopsies. PTHrP immunoreactivity and PTHrP gene expression are present in sarcoid granulomata. PTHrP may contribute to the hypercalcemia of sarcoidosis.