Biomedical subjects
T J Martin
Publications and source records attributed to T J Martin.
Nuclear and nucleolar localization of parathyroid hormone-related protein.
Parathyroid hormone-related protein (PTHrP) was first discovered as the factor causing hypercalcaemia produced by solid tumours frequently associated with the head and neck, breast, lung and kidney. The homology of its amino-terminus to parathyroid hormone (PTH; eight of the first 13 residues are identical), enables it to share the same receptor and perform similar biological functions to PTH. The sequences of PTHrP C-terminal to its PTH-like region confer functions such as transplacental calcium transport, renal bicarbonate excretion and in vitro osteoclast inhibition. Recent findings have shown that PTHrP is a nuclear/nucleolar protein in certain tissues and that this localization is cell cycle-regulated, mediated by the middle portion of the molecule, and involves the nuclear import receptor importin beta1. The present review discusses what is known about the pathway by which PTHrP localizes to the nucleus/nucleolus and the putative roles it may have there.
Abnormalities of eyelid position and function.
Evaluation of the eyelids is an important part of the neuro-ophthalmic examination. Abnormal eyelid position and function can be caused by disorders involving the third cranial nerve, the oculosympathetic pathway, and the seventh cranial nerve, as well as supranuclear pathways, or as a result of neuromuscular diseases. To avoid unwarranted neurological investigations, it is also important for the clinician to recognize non-neurological eyelid abnormalities (such as ptosis from levator dehiscence or eyelid edema).
A randomized, prospective evaluation of noninvasive ventilation for acute respiratory failure.
We compared noninvasive positive-pressure ventilation (NPPV), using bilevel positive airway pressure, with usual medical care (UMC) in the therapy of patients with acute respiratory failure (ARF) in a prospective, randomized trial. Patients were subgrouped according to the disease leading to ARF (chronic obstructive pulmonary disease [COPD], a non-COPD-related pulmonary process, neuromuscular disease, and status postextubation), and were then randomized to NPPV or UMC. Thirty-two patients were evaluated in the NPPV group and 29 in the UMC group. The rate of endotracheal intubation (ETI) was significantly lower in the NPPV than in the UMC group (6.38 intubations versus 21.25 intubations per 100 ICU days, p = 0.002). Mortality rates in the intensive care unit (ICU) were similar for the two treatment groups (2.39 deaths versus 4.27 deaths per 100 ICU days, p = 0.21, NPPV versus UMC, respectively). Patients with hypoxemic ARF in the NPPV group had a significantly lower ETI rate than those in the UMC group (7.46 intubations versus 22.64 intubations per 100 ICU days, p = 0.026); a similar trend was noted for patients with hypercapnic ARF (5.41 intubations versus 18.52 intubations per 100 ICU days, p = 0.064, NPPV versus UMC, respectively). Patients with ARF in the non-COPD category had a lower rate of ETI with NPPV than with UMC (8.45 intubations versus 30.30 intubations per 100 ICU days, p = 0.01). Although the rate of ETI was lower among COPD patients receiving NPPV, this trend did not reach statistical significance (5.26 intubations versus 15.63 intubations per 100 ICU days, p = 0.12, NPPV versus UMC, respectively). In conclusion, NPPV with bilevel positive airway pressure reduces the rate of ETI in patients with ARF of various etiologies.
Intracellular calcium and protein kinase C mediate expression of receptor activator of nuclear factor-kappaB ligand and osteoprotegerin in osteoblasts.
Receptor activator of nuclear factor-kappaB ligand (RANKL) and osteoprotegerin (OPG) produced by osteoblasts/stromal cells are involved as positive and negative regulators in osteoclast formation. Three independent signals have been proposed to induce RANKL expression in osteoblasts/stromal cells: vitamin D receptor-, cAMP-, and gp130-mediated signals. We previously reported that intracellular calcium-elevating compounds such as ionomycin, cyclopiazonic acid, and thapsigargin induced osteoclast formation in cocultures of mouse bone marrow cells and primary osteoblasts. Increases in calcium concentration in culture medium also induced osteoclast formation in cocultures. Treatment of primary osteoblasts with these compounds or with high calcium medium stimulated the expression of both RANKL and OPG messenger RNAs (mRNAs). 1,2-Bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid)-tetra(acetoxymethyl)ester, an intracellular calcium chelator, suppressed both ionomycin-induced osteoclast formation in cocultures and expression of RANKL and OPG mRNAs in primary osteoblasts. Phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C, also stimulated osteoclast formation in these cocultures and the expression of RANKL and OPG mRNAs in primary osteoblasts. Protein kinase C inhibitors such as calphostin and staurosporin suppressed ionomycin- and PMA-induced osteoclast formation in cocultures and expression of RANKL and OPG mRNAs in primary osteoblasts. Ionomycin stimulated RANKL mRNA expression in ST2 and MC3T3-G2/PA6 cells, but not in MC3T3-E1 or NIH-3T3 cells. These effects were closely correlated with osteoclast formation in response to ionomycin in cocultures with these stromal cell lines. OPG strongly inhibited osteoclast formation induced by calcium-elevating compounds and PMA in cocultures, suggesting that RANKL expression in osteoblasts is a rate-limiting step for osteoclast induction. Forskolin, an activator of cAMP signals, also stimulated osteoclast formation in cocultures. Forskolin enhanced RANKL mRNA expression but suppressed OPG mRNA expression in primary osteoblasts. These results suggest that the calcium/protein kinase C signal in osteoblasts/stromal cells is the fourth signal for inducing RANKL mRNA expression, which, in turn, stimulates osteoclast formation.
Osteoprotegerin produced by osteoblasts is an important regulator in osteoclast development and function.
Osteoprotegerin (OPG), a soluble decoy receptor for receptor activator of nuclear factor-kappaB ligand (RANKL)/osteoclast differentiation factor, inhibits both differentiation and function of osteoclasts. We previously reported that OPG-deficient mice exhibited severe osteoporosis caused by enhanced osteoclastic bone resorption. In the present study, potential roles of OPG in osteoclast differentiation were examined using a mouse coculture system of calvarial osteoblasts and bone marrow cells prepared from OPG-deficient mice. In the absence of bone-resorbing factors, no osteoclasts were formed in cocultures of wild-type (+/+) or heterozygous (+/-) mouse-derived osteoblasts with bone marrow cells prepared from homozygous (-/-) mice. In contrast, homozygous (-/-) mouse-derived osteoblasts strongly supported osteoclast formation in the cocultures with homozygous (-/-) bone marrow cells, even in the absence of bone-resorbing factors. Addition of OPG to the cocultures with osteoblasts and bone marrow cells derived from homozygous (-/-) mice completely inhibited spontaneously occurring osteoclast formation. Adding 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] to these cocultures significantly enhanced osteoclast differentiation. In addition, bone-resorbing activity in organ cultures of fetal long bones derived from homozygous (-/-) mice was markedly increased, irrespective of the presence and absence of bone-resorbing factors, in comparison with that from wild-type (+/+) mice. Osteoblasts prepared from homozygous (-/-), heterozygous (+/-), and wild-type (+/+) mice constitutively expressed similar levels of RANKL messenger RNA, which were equally increased by the treatment with 1alpha,25(OH)2D3. When homozygous (-/-) mouse-derived osteoblasts and hemopoietic cells were cocultured, but direct contact between them was prevented, no osteoclasts were formed, even in the presence of 1alpha,25(OH)2D3 and macrophage colony-stimulating factor. These findings suggest that OPG produced by osteoblasts/stromal cells is a physiologically important regulator in osteoclast differentiation and function and that RANKL expressed by osteoblasts functions as a membrane-associated form.
In vivo demonstration that human parathyroid hormone 1-38 inhibits the expression of osteoprotegerin in bone with the kinetics of an immediate early gene.
Osteoprotegerin (OPG) is a potent inhibitor of osteoclast formation and function. To elucidate how OPG is regulated in bone, we examined (1) the expression and localization of OPG protein in bone tissue, (2) the effect of human parathyroid hormone 1-38 (hPTH 1-38) on OPG messenger RNA (mRNA) levels in rat femur metaphyseal and diaphyseal bone, and (3) the effect of hPTH(1-38) on expression of OPG mRNA in cultured osteoblast-like cells derived from the metaphysis and diaphysis, and in ROS 17/2.8 osteosarcoma cells. Because PTH has been shown to stimulate osteoblast activity via the cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) signal transduction pathway we also investigated whether PTH action on OPG in vivo is dependent on activation of cAMP/PKA pathway. Immunohistochemistry was used to evaluate OPG protein expression and Northern blot hybridization was used to analyze OPG mRNA expression both in vivo and in vitro. Immunohistochemistry of OPG protein expression in the rat distal femur metaphysis revealed that it was localized predominantly in preosteoblasts, osteoblasts, lining cells, and the osteoid layer, with occasional immunoreactivity in osteocytes and cells of the bone marrow. Subcutaneous (sc) administration of a single injection of hPTH(1-38) at 80 microg/kg induced a rapid and transient decrease in OPG mRNA expression in both metaphyseal and diaphyseal bone. The decrease in OPG message was evident by 1 h and mRNA levels returned to baseline after 3 h. PTH analog PTH(1-31), which stimulates intracellular cAMP accumulation, inhibited OPG expression, whereas PTH analogs (3-34 and 7-34) that do not stimulate cAMP production had no effect on expression. In contrast to PTH, prostaglandin E2 (PGE2) had no effect on OPG mRNA expression in vivo in the metaphyseal bone cells, under conditions in which PGE2 does promote expression of the c-fos gene. The in vivo effects of hPTH(1-38) on OPG mRNA were confirmed in isolated primary osteoblast cultures derived from either metaphyseal or diaphyseal bone as well as in ROS 17/2.8 osteosarcoma cells. We propose that the rapid and transient decrease in OPG expression may initiate a cascade of events resulting in the differentiation of osteoclast progenitor. Such a spatially and temporally programmed effect of PTH might contribute to bone turnover.
Fibroblastic stromal cells express receptor activator of NF-kappa B ligand and support osteoclast differentiation.
Osteoclast formation in bone is supported by osteoblasts expressing receptor activator of NF-kappa B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) expression. Numerous osteotropic factors regulate expression levels of RANKL and the RANKL decoy receptor osteoprotegerin (OPG) in osteoblasts, thereby affecting osteoclast differentiation. However, not only in RANKL widely expressed in soft tissues, but osteoclasts have been noted in extraskeletal lesions. We found that cultured skin fibroblastic cells express RANKL, M-CSF, and OPG messenger (mRNA). Stimulation by 1 alpha,25 dihydroxyvitamin D3 [1,25(OH)2D3] plus dexamethasone (Dex) augmented RANKL and diminished OPG mRNA expression in fibroblastic cells and caused the formation of numerous osteoclasts in cocultures of skin fibroblastic cells with hemopoietic cells or monocytes. The osteoclasts thus formed expressed tartrate-resistant acid phosphatase (TRAP) and calcitonin (CT) receptors and formed resorption pits in cortical bone. Osteoclast formation also was stimulated (in the presence of Dex) by prostaglandin E2 (PGE2), interleukin-11 (IL-11), IL-1, tumor necrosis factor-alpha (TNF-alpha), and parathyroid hormone-related protein (PTHrP), factors which also stimulate osteoclast formation supported by osteoblasts. In addition, granulocyte-macrophage-CSF (GM-CSF), transforming growth factor-beta (TGF-beta), and OPG inhibited osteoclast formation in skin fibroblastic cell-hemopoietic cell cocultures; CT reduced only osteoclast nuclearity. Fibroblastic stromal cells from other tissues (lung, respiratory diaphragm, spleen, and tumor) also supported osteoclast formation. Thus, RANKL-positive fibroblastic cells in extraskeletal tissues can support osteoclastogenesis if osteolytic factors and osteoclast precursors are present. Such mesenchymally derived cells may play a role in pathological osteolysis and may be involved in osteoclast formation in extraskeletal tissues.
Mechanisms in the skeletal complications of breast cancer.
Breast cancer is the most common malignancy in women, with a worldwide prevalence of 1.5 million throughout the industrialized countries. Its mortality rate is second only to lung cancer in the USA and Europe. Its high incidence and prevalence makes it a major public health problem. Up to one-third of women with early stage breast cancer will eventually succumb to the disease, and most of these will develop bone metastases during the course of the disease. Approximately 70% of patients with breast cancer have bone metastases, with 27% having lung and liver metastases. Hypercalcemia also is very common in advanced breast cancer, manifesting itself in one-third of patients late in the disease. Breast cancer accounts for approximately 25% of the cases of hypercalcemia in cancer. The major skeletal complications of breast cancer--hypercalcemia and bone metastases--almost certainly share certain mechanisms and these will be discussed.
Antagonism of delta(2)-opioid receptors by naltrindole-5'-isothiocyanate attenuates heroin self-administration but not antinociception in rats.
delta-Opioid receptors have been implicated in reinforcement processes and antagonists are available that produce long-lasting and selective antagonism of delta-opioid receptors in vivo. This experiment assessed the contribution of delta-opioid receptors to the antinociceptive and reinforcing properties of heroin. The effects of the irreversible delta-antagonist naltrindole-5'-isothiocyanate (5'-NTII) were evaluated on heroin self-administration and hot-plate antinociception in rats. 5'-NTII (10 nmol i.c.v.) shifted the dose-response curve for heroin self-administration downward, increasing the A(50) values on the ascending and descending limbs by approximately 0.5 log units and decreasing the maximum by 33%. 5'-NTII (40 nmol i.c.v.) shifted both limbs of the heroin self-administration dose-effect curve 1.2 log units to the right and decreased the maximum by 90%. Heroin self-administration gradually returned to baseline levels over 7 or 17 days after administration of 10 or 40 nmol 5'-NTII, respectively. 5'-NTII (40 nmol i.c.v.) decreased the self-administration of 0.17 mg/infusion cocaine by 40% while having no effect on responding maintained by 0.33 or 0.67 mg/infusion. 5'-NTII attenuated the antinociceptive effects of deltorphin (delta(2)) in a dose-dependent manner while having no effect on antinociception elicited after i.c. v. administration of [D-Pen(2),D-Pen(5)]-enkephalin (delta(1)) or [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin (mu). In addition, the antinociceptive effects of heroin were not significantly affected by 5'-NTII (40 nmol i.c.v.). Therefore, 5'-NTII can attenuate the reinforcing effects of heroin at doses that do not affect its antinociceptive effects. Long-acting delta(2)-opioid antagonists may be beneficial in the treatment of heroin dependence or as adjuncts to reduce the abuse liability of opioid analgesics.
Parathyroid hormone-related protein (PTHrP) in cartilaginous and bony fish tissues.
Tissues from a range of fish were examined for the presence of parathyroid hormone-related protein (PTHrP) to investigate PTHrP protein distribution and PTHrP gene expression in jawless fish, cartilaginous fish, and bony fish. Immunoreactive PTHrP was localized using antisera to N-terminal and mid-molecule regions of human PTHrP and PTHrP gene expression examined using a digoxigenin labeled riboprobe to a conserved region of the mammalian PTHrP gene. In all of the fish studied, PTHrP protein and messenger RNA (mRNA) were localized to the skin, kidney, and skeletal muscle, following the pattern seen in higher vertebrates. Additional sites of localization for both protein and mRNA included gill, nerve cord, and pituitary, as well as developing dermal denticles and rectal gland in the elasmobranch species. The sites of PTHrP distribution indicate that PTHrP may have roles in ionoregulation as well as growth and differentiation in fish, as has been suggested in higher vertebrates. The results imply that the distribution of PTHrP is widespread in fish and that there is homology between the PTHrP molecules found in humans and fish. The conservation of localization and possible similarity of the PTHrP molecules between tetrapods and fish suggests that PTHrP has a number of fundamental roles in vertebrates. J. Exp. Zool. 284:541-548, 1999.
Phosphorylation at the cyclin-dependent kinases site (Thr85) of parathyroid hormone-related protein negatively regulates its nuclear localization.
Parathyroid hormone-related protein (PTHrP) is expressed by a wide variety of cells and is considered to act as a secreted factor; however, evidence is accumulating for it to act in an intracrine manner. We have determined that PTHrP localizes to the nucleus at the G1 phase of the cell cycle and is transported to the cytoplasm when cells divide. PTHrP contains a putative nuclear localization sequence (NLS) (residues 61-94) similar to that of SV40 T-antigen, which may be implicated in the nuclear import of the molecule. We identified that Thr85 immediately prior to the NLS of PTHrP was phosphorylated by CDC2-CDK2 and phosphorylation was cell cycle-dependent. Mutation of Thr85 to Ala85 resulted in nuclear accumulation of PTHrP, while mutation to Glu85 to mimic a phosphorylated residue resulted in localization of PTHrP to the cytoplasm. Combined, the data demonstrate that the intracellular localization of PTHrP is phosphorylation- and cell cycle-dependent, and such control further supports a potential intracellular role (10,34,35) for PTHrP.
Importin beta recognizes parathyroid hormone-related protein with high affinity and mediates its nuclear import in the absence of importin alpha.
Parathyroid hormone-related protein (PTHrP), expressed in a range of tumors, has endocrine, autocrine/paracrine, and intracrine actions, some of which relate to its ability to localize in the nucleus. Here we show for the first time that extracellularly added human PTHrP (amino acids 1-108) can be taken up specifically by receptor-expressing UMR106.01 osteogenic sarcoma cells and accumulate to quite high levels in the nucleus and nucleolus within 40 min. Quantitation of recognition by the nuclear localization sequence (NLS)-binding importin subunits indicated that in contrast to proteins containing conventional NLSs, PTHrP is recognized exclusively by importin beta and not by importin alpha. The sequence of PTHrP responsible for binding was mapped to amino acids 66-94, which includes an SV40 large tumor-antigen NLS-like sequence, although sequence determinants amino-terminal to this region were also necessary for high affinity binding (apparent dissociation constant of approximately 2 nM for importin beta). Nuclear import of PTHrP was assessed in vitro using purified components, demonstrating that importin beta, together with the GTP-binding protein Ran, was able to mediate efficient nuclear accumulation in the absence of importin alpha, whereas the addition of nuclear transport factor NTF2 reduced transport. The polypeptide ligand PTHrP thus appears to be accumulated in the nucleus/nucleolus through a novel, NLS-dependent nuclear import pathway independent of importin alpha and perhaps also of NTF2.
Dual posttranscriptional targets of retinoic acid-induced gene expression.
Retinoic acid-induced differentiation of the pre-osteoblastic cell line, UMR 201, is associated with a marked increase in the proficiency of posttranscriptional nuclear processing of alkaline phosphatase mRNA. In this study we attempted to correlate the posttranscriptional actions of retinoic acid with changes in phosphorylation, or abundance of spliceosome components, or both. Treatment with retinoic acid for periods of < or = 4 h resulted in dephosphorylation of nuclear U1 70K protein without affecting its abundance. Peptide mapping showed that U1 70K dephosphorylation was related to the disappearance of one specific phosphopeptide out of four major U1 70K phosphopeptides. A twofold decrease in mRNA expression of an isoform of alternative splicing factor that inhibits splicing was also observed over the same period. Tumor necrosis factor-alpha, which enhances the posttranscriptional action of retinoic acid, reduced U1 70K mRNA expression, while an inhibition of retinoic acid action by transforming growth factor-beta was associated with a marked increase in U1 70K mRNA levels. Our results draw attention to the complex interactions between short- and long-term alterations in the abundance and functional status of U1 70K, as well as SR proteins by growth and/or differentiation factors in the regulation of spliceosome formation and function.
Activated T lymphocytes support osteoclast formation in vitro.
Osteoblastic stromal cells are capable of supporting osteoclast formation from hematopoietic precursors in the presence of osteotropic factors such as 1alpha,25(OH)(2)D(3), PTH, and IL-11. Osteoblastic stromal cells produce receptor activator of NF-kappaB ligand (RANKL), a type II membrane protein of the TNF ligand family, in response to these agents. Activated T lymphocytes also produce RANKL; however, the ability of this cell type to support osteoclast formation in vitro is unknown. Human PBMC-derived T cells, extracted using alphaCD3-coated magnetic beads, were cocultured with adherent murine spleen cells in the presence of Con A and a panel of cytokines. In the presence of Con A, bona fide osteoclasts were formed in vitro with activated T cells: IL-1alpha and TGFbeta further enhanced osteoclast numbers. PBMC-derived lymphocytes showed an increase in the mRNA expression of RANKL within 24 h of treatment with the same agents that were used to induce osteoclast formation. In synovial tissue sections with lymphoid infiltrates from RA patients, the expression of RANKL was demonstrated in CD3(+) T cells. The ability of activated T lymphocytes to support osteoclast formation may provide a mechanism for the potentiation of osteoclast formation and bone resorption in disease states such as rheumatoid arthritis.
Age- and gender-specific rate of fractures in Australia: a population-based study.
There is little population-based data concerning fracture rates in Australia. We ascertained all fractures occurring during 2 years in adults aged 35 years and over residing within a defined region (population 218 000), representative of the Australian population. The major strength of this study is the comprehensive ascertainment of fractures, which was ensured by regular searches of the only two radiologic providers in the Geelong Osteoporosis Study region. Nevertheless, vertebral fractures are likely to be underestimated since our ascertainment relied on a clinical indication for a medical imaging procedure. Among those aged 35-55 years, the fracture rate (persons per 10,000/year) in men was about double the rate in women (65 vs 35). The fracture rate was almost 7 times higher in women over 60 years versus women less than 55 years of age. In contrast, the fracture rate in men over 60 years was only 50% higher than in men less than 55 years of age (72 vs 104). Fracture rates in women and men were highest at the hip (28 and 10 respectively), spine (21 and 7), distal forearm (Colles') (18 and 4) and humerus (11 and 3), and were 3-4 times higher in women than men. These fractures accounted for 63% of all fractures in women and 32% in men. By contrast, the rate of lower leg and ankle fractures was less than 10 per 10,000 in both women and men and did not increase to the same extent with age. Hip fracture rates appear high, particularly among the older age strata, compared with retrospective ascertainment in other populations. In Australia, as in many other countries, there is an increasing longevity of the population. The number of women aged 90 years and over increased by 32% and the number of men of this age increased by 48% in the 5 years between the Australian national census of 1991 and 1996. Given stable fracture rates, the substantial health burden imposed by age-related fractures, particularly hip fractures, will continue to escalate in both women and men.
Interim report and recommendations of the World Health Organization Task-Force for Osteoporosis.
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