Munmun Chakraborty

CELLULAR AND MOLECULAR BIOLOGY OF THE OSTEOCLAST

Publisher Summary This chapter discusses the cellular and molecular biology of the osteoclast. The osteoclast is the cell responsible for the resorption of the bone matrix. Bone resorption is a necessary process for the normal development of the skeleton, for its adaptability, and for its maintenance. This cellular process is essential in the growth, remodeling, and repair of bone and is, under normal conditions, tightly coupled to the process of bone formation by the osteoblast. It is the balance between these two cellular activities that determines skeletal mass and shape at any point in time. The main determinants of the biology of the osteoclast are first its attachment to the bone matrix, leading to the formation of the sealed-off bone resorbing compartment and, second, the polarized acidification of, and secretion of enzymes into, this compartment. These activities require tight control of different membrane domains both in terms of their composition and targeting and in terms of ion transport. The biology and activity of these membrane domains are strongly interdependent.

Signal transduction by calcitonin Multiple ligands, receptors, and signaling pathways.

Calcitonin (CT) is a peptide hormone that is secreted by the parafollicular cells of the thyroid in response to elevated serum calcium levels. It acts to reduce serum calcium by inhibiting bone resorption and promoting renal calcium excretion. In addition to this hypocalcemie effect, calcitonin modulates the renal transport of water and several ions other than calcium and acts on the central nervous system to induce analgesia, anorexia, and gastric secretion. The CT receptor, a member of a newly described family of serpentine G protein-coupled receptors, has recently been shown to couple to multiple trimeric G proteins, thereby activating several signaling proteins, including protein kinase C, cAMP-dependent protein kinase and calcium/calmodulin-dependent protein kinase. In kidney proximal tubule cells (LLC-PK1), the CT-activated signaling mechanisms vary in a cell cycle-dependent manner, with the receptor coupling through a G(s) protein during G(2) phase and through a G(i) protein and possibly a G(q) protein during S phase. These signaling mechanisms differentially modulate the activities of Na(+)/K(+)-ATPase and the apical Na(+)/H(+) exchanger, effector molecules that play important roles in transepithelial Na(+) transport. Cloning of CT receptors has revealed the presence of alternatively spliced cassettes, resulting in the expression of different isoforms of the receptor. The availability of these recombinant CT receptors has allowed preliminary characterization of the effects of changes in the receptors structure on its ligand binding and signal transduction properties. Thus, the cellular and molecular biology of CT is complex, with several structurally related peptide ligands and multiple isoforms of the CT receptor that can independently activate diverse signaling pathways. As the recent exciting results in this field are extended, we can expect rapid progress in understanding the molecular basis of the diverse effects of CT and, possibly, of the CT-related peptides CGRP and amylin.

Biology of the Osteoclast

The osteoclast is the cell responsible for the resorption of the extracellular bone matrix. Under physiological conditions, bone resorption plays an essential role in the homeostasis of both the skeleton and serum calcium. This cellular process is also essential in the growth and remodeling of bone, where it is tightly coupled to the process of bone formation by the osteoblast. It is the integrated functions of these two cell types that lead to the quantitative and qualitative maintenance of the skeleton, to the changes in size and shape of the individual bones during growth, and to bone repair after trauma or fracture. On the other hand, it is the disruption of the coupling between bone resorption and formation that leads to abnormally dense (osteopetrosis and osteosclerosis) or porous bone (osteoporosis). A coupled but high resorption rate characterizes high bone-turnover diseases, such as hyperparathyroidism or Paget’s disease, and leads to the disruption of the architecture and function of the skeleton.

Thyroidal influence on the cell surface GM1 of granule cells: Its significance in cell migration during rat brain development

Summary1.No difference was observed in thein vitro growing ability of granule cells isolated from hypothyroid or normal rat brain. When granule cells were taken from hypothyroid rat brain and grown in normal culture medium containing 10% fetal calf serum, they behaved similarly to the granule cells obtained from normal rat brain.2.In both cases there were progressive losses ofin vitro growing ability of the granule cells with the age of the animal and it became impossible to grow them when derived from 21 days or older animals.3.A marked decrease in cell surface GM1 was observed when the cells were maintained under thyroid hormone-deficient conditions in culture.4.Anti-GM1 antibody was found to inhibit significantly the migration of granule cells along the astrocyte fibers.5.These results indicate that GM1 has an important role in thyroid hormone-dependent postnatal brain maturation in rat.

Human platelet dense granules: improved isolation and preliminary characterization of [3H]-serotonin uptake and tetrabenazine-displaceable [3H]-ketanserin binding.

An improved method for the isolation of human platelet dense granules was developed. A good yield (45%) of highly enriched (69-fold, based on serotonin content) dense granules was obtained after mild sonication and Percoll gradient centrifugation. The method has facilitated characterization of the granule, permitting the first report of Km and Vmax values for [3H]-serotonin uptake, as well as the first determination of Kd and Bmax values for tetrabenazine-displaceable [3H]-ketanserin binding, in the human platelet dense granule. The rates and affinities (Vmax 1.45 nmol/mg/min, Km 0.93 uM) of [3H]-serotonin uptake were similar to those previously reported for porcine dense granules. Tetrabenazine-displaceable [3H]-ketanserin binding was observed with a Kd (9.4 nM) similar to, and a Bmax (5.4 pmol/mg) approximately 10-fold lower than, that previously seen in bovine chromaffin granules.

Use of high-performance liquid chromatography for assay of glutamic acid decarboxylase. Its limitation in use for post-mortem brain.

Rat brain, obtained 10 min after death, contained high levels of endogenous gamma-aminobutyric acid (GABA) and glutamic acid. Incubation of this brain homogenate at 37 degrees C indicated decrease of GABA with time due to degradation by GABA-transaminase. Reported high-performance liquid chromatographic (HPLC) methods for glutamic acid decarboxylase (GAD) assay depend on the difference between the GABA content of the reaction mixture after and before the incubation period. None of the methods considered the degradation of GABA during incubation. Furthermore, during determination of the Michaelis constant (KM) for the reaction none of them considered the endogenous substrate. Here we have focused on these factors which seriously affect the maximum velocity (Vmax) and KM values during GAD assay by the HPLC technique. By a simple and rapid HPLC technique we have measured GAD activity in post-mortem rat brain after removing endogenous glutamic acid by charcoal treatment and using gabaquline to prevent GABA degradation during incubation period. By this method a Vmax value of 46 +/- 4 nmol/h/mg protein and a KM value of 7.5 +/- 0.6 mM were observed for GAD activity of crude brain homogenate. For a comparative study, we have carried out radiometric assay of GAD activity from the same sample and observed a Vmax of 48 +/- 6 nmol/h/mg protein and KM of 6.9 +/- 0.4 mM.

Coupling of Nerve Growth Factor to Its Receptor: Inhibition by Anti-GM3 Ganglioside Antibody

Abstract1. Normal differentiation of PC 12 cells and dorsal root ganglionic neurons in culture need nerve growth factor (NGF) for their neurite outgrowth.2. An antibody against GM3 ganglioside was found to inhibit the nerve growth factor mediated neurite formation of both the cells in vitro significantly.3. Further analysis revealed that the binding of 125I-NGF to live PC 12 cells could be markedly inhibited by anti-GM3 antibody in a dose dependent manner.4. Scatchard analysis revealed that in the presence of anti-GM3 antibody only some low affinity binding sites were available for NGF—high affinity binding sites were totally blocked.5. These results further strengthen the hypothesis that anti-GM3 antibody affects neuronal cell growth by interfering with the coupling of growth factors to their cell surface receptors.

1,25‐dihydroxyvitamin D3 selectively induces increased expression of the Na,K‐ATPase β1 subunit in avian myelomonocytic cells without a concomitant change in Na,K‐ATPase activity

Treatment of avian myelomonocytic cells with 1,25‐dihydroxyvitamin D3 (1,25(OH)2D3) results in an approximately two fold increase in levels of Na,K‐ATPase β1 subunit mRNA and protein (both total and plasma membrane‐associated). The changes in β1 subunit expression occur in the absence of a detectable increase in expression of any of the three α subunit isoforms or in Na,K‐ATPase activity. The selective induction of the expression of the β subunit in avian myelomonocytic cells by 1,25(OH)2D3 reveals a previously unobserved feature of the regulation of Na,K‐ATPase expression, while the targeting of β subunit polypeptides to the plasma membrane in the absence of a corresponding increase in active Na,K‐ATPase suggests that, in these cells, transport of the β subunit to the plasma membrane may be independent of its binding to the α subunit. J. Cell. Physiol. 172:221–229, 1997.