Characterization of circulating human osteoclast progenitors: development of in vitro resorption assay.

Several cell surface markers were used to isolate monocytes as osteoclast progenitors with an immunomagnetic cell separation system. Use of this system with specific monocyte antibodies produced 99% pure monocytes. When purified monocytes were cultured on bovine bone slices in the presence of receptor activator of nuclear factor-kappaB (RANKL), macrophage-colony stimulating factor (M-CSF), tumor necrosis factor alpha (TNF-alpha), and dexamethasone for 14 days, CD14(+) CD11b(+), and CD61(+) monocytes had approximately 90-, 30- and 20-fold higher osteoclast formation capacities/plated cells compared to the control culture. CD15(+) monocytes generated few tartrate-resistant acid phosphatase-positive multinucleated cells (TRACP+ MNC), and CD169(+) monocytes generated no TRACP+ MNC. This suggests, that there are various subsets of monocytes in the blood circulation and that they have different capacities in osteoclast formation. These results show that circulating human osteoclast progenitors can be efficiently purified by immunomagnetic cell separation system using anti-CD14, -CD11b, and -CD61 antibodies. These purified monocyte fractions had different ability to give rise to osteoclasts. CD169 was not found to be suitable for osteoclast progenitor isolation. Optimal concentration of dexamethasone for osteoclast formation and bone resorption was 10 nM. To develop a human resorption assay, osteoclasts were first induced for 7 days, whole media were replaced, cultures were continued for additional 3 days and C-terminal telopeptide of type I collagen was determined from culture media. This assay was shown to be functional, since two well-known resorption inhibitors, bafilomycin A(1) and calcitonin, dose-dependently inhibited the resorption activity of osteoclasts.

Translocation of MARCKS and reorganization of the cytoskeleton by PMA correlates with the ion selectivity, the confluence, and transformation state of kidney epithelial cell lines.

The role of protein kinase C (PKC) in the regulation of the cytoskeleton of epithelial cells with tightly sealed contacts, poor contacts, and without contacts were investigated by incubating them with a protein kinase C activator phorbol myristoyl acetate (PMA). The morphology and organization of the membrane skeleton and stress fibers as well as the localization of an actin-bundling PKC substrate MARCKS in confluent MDCK cells originating from the distal tubulus of dog kidney, LLC-PK1 cells originating from the proximal tubulus of pig kidney, src-transformed MDCK cells, epidermoid carcinoma A431 cells, and MDCK cells grown in low calcium medium (LC medium) in low density were visualized with phase contrast and immunofluorescence microscopy. Four different responses to the PMA-treatment in actin-based structures of cultured epithelial cells were observed: 1) disintegration of the membrane skeleton in confluent MDCK cells; 2) depolymerization of the stress fibers in confluent MDCK and LLC-PK1 cells; 3) formation of the membrane skeleton in A431 cells, and 4) formation of the stress fibers and membrane skeleton in LC-MDCK cells. Thus, it seems that in fully confluent tightly sealed epithelium, activation of PKC has a deleterious effect on actin-based structures, whereas in cells without contacts or loose contacts, activation of PKC by PMA results in improvement of actin-based cytoskeletal structures. The main difference between the two kidney cell lines used is their selectivity to ion transport: the monolayer of LLC-PK1 cells is anion selective and MDCK cells cation selective. We propose a model where alterations in the ionic milieu within the MDCK cells by means of cation channels affect the disintegration of the membrane skeleton. The distribution of MARCKS followed the distribution of fodrin in both cell lines upon PMA-treatment, suggesting that phosphorylation of MARCKS by PKC may contribute in the regulation of the integrity of the membrane skeleton.

Immunolocalization of the fodrin, E-cadherin, and beta-catenin adhesion complex in infiltrating ductal carcinoma of the breast-comparison with an in vitro model.

Fodrin, E-cadherin, and beta-catenin immunolocalization was studied in 54 cases of infiltrating ductal carcinoma of the breast and compared with an in vitro model in order to study the dynamic relationship between these components of an adhesion complex. In low-grade tumours, the staining patterns were similar for both fodrin and E-cadherin, with localization of these proteins to the cell membranes. beta-Catenin showed reduced membrane staining compared with non-neoplastic epithelium. High-grade tumours displayed strong membranous as well as cytoplasmic immunolocalization of fodrin, while E-cadherin staining was fragmented or lost from the membranes, with only occasional weak intracellular staining. beta-Catenin showed fragmented membrane staining and cytoplasmic accumulation. In addition, nuclear staining of beta-catenin was occasionally observed. In a v-src-transformed MDCK cell line, following 15min of src activation, beta-catenin began to detach from the cell membrane and localize to the cytoplasm, while fodrin and E-cadherin remained unchanged. After 30-45min of src activation, the cells lost their cuboidal shape and began to lose cell-to-cell contact. Fodrin staining remained mostly membranous while that of E-cadherin and beta-catenin was fragmented and spiky. After 60min of src activation, fodrin localized completely in the cell cytoplasm, while E-cadherin and beta-catenin were partly cytoplasmic with fragmented and spiky membranous staining. Occasionally, beta-catenin was seen in the nucleus. Both in vivo and in vitro findings clearly demonstrated a disruption of the E-cadherin/beta-catenin/fodrin/cytoskeleton linkage concomitant with the loss of cell-to-cell adhesion and change in cell shape, from epithelioid to a fibroblastoid phenotype. Membranous localization of E-cadherin showed a positive correlation with oestrogen and progesterone expression, whereas loss of membranous E-cadherin and cytoplasmic accumulation of fodrin was more often observed in high-grade carcinomas and showed a positive correlation with p53 expression.

Effect of PMA on the integrity of the membrane skeleton and morphology of epithelial MDCK cells is dependent on the activity of amiloride-sensitive ion transporters and membrane potential.

The signaling pathways from an activation of protein kinase C (PKC) by phorbol myristate acetate (PMA) to the rearrangement of actin-based cytoskeleton and membrane skeleton of epithelial MDCK cells were studied by visualizing the cytoskeletal organization with immunofluorescence microscopy and by measuring intracellular pH, sodium ion concentration and membrane potential with the aid of fluorescent intracellular indicators. Upon PMA treatment the MDCK cells lost their cubic shape and acquired a spindle-like morphology. The stress fibers were depolymerized, and fodrin, the main component of the membrane skeleton, was released from the lateral walls to the cytosol. These changes were accompanied by depolarization of the cells, decrease in the intracellular pH and sodium ion concentration. In order to test the mutual correlation between the PMA-induced alterations we treated the cells with PMA in the presence of channel inhibitors or ionophores and in defined media. The effects of PMA on the membrane skeleton and morphology could be reversed in media lacking Na+ or K+ ions or by hyperpolarizing agents, dimethylamiloride and valinomycin, suggesting that the effects of PMA on the cytoskeleton were dependent on the ion gradients and membrane potential across the cell membrane. Moreover, the morphological changes and instabilization of the membrane skeleton of MDCK cells took place spontaneously without PMA in depolarizing conditions, in potassium gluconate buffer. We suggest that the membrane potential across the cell membrane of MDCK cells together with the activity of amiloride-sensitive cation transporters transmits signals in the protein kinase C (PKC) pathway leading from activation of PKC to fibroblast-like morphology and cytoplasmic localization of membrane skeleton components, features characteristic for cancer cells.

Regulation of the disassembly/assembly of the membrane skeleton in Madin-Darby canine kidney cells.

The effects of pH, temperature, block of energy production, calcium/calmodulin, protein phosphorylation, and cytoskeleton-disrupting agents (cytochalasin D, nocodazole) on the integrity of the membrane skeleton were studied in polarized MDCK cells. The intracellular distributions of alpha-fodrin, actin, and ankyrin were monitored by immunofluorescence microscopy. The membrane skeleton, once assembled, seemed to be quite stable; the only factors releasing alpha-fodrin from the lateral walls were the acidification of the cytoplasm and the depletion of extracellular calcium ions. Upon cellular acidification, some actin was also released from its normal location along the lateral walls and was seen in colocalization with alpha-fodrin in the cytoplasm, whereas ankyrin remained associated with the lateral walls. No accumulation of plasma membrane lipids was observed in the cytoplasm of acidified cells, as visualized by TMA-DPH. These results suggest that the linkages between the fodrin-actin complex and its membrane association sites are broken upon acidification. The pH-induced change in alpha-fodrin localization was reversible upon restoring the normal pH. Reassembly of the membrane skeleton, however, required temperatures above +20 degrees C, normal energy production, proper cell-cell contacts, and polymerized actin. Release of alpha-fodrin from the lateral walls to the cytoplasm was also observed upon depletion of extracellular calcium ions. This change was accompanied by the disruption of cell-cell contacts, supporting the role of proper cell-cell contacts in the maintenance of the membrane skeleton polarity. These results suggest that local alterations of the cytoplasmic pH and calcium ion concentration may be important in regulating the integrity of the membrane skeleton.

The effects of PMA and TFP and alterations in intracellular pH and calcium concentration on the membrane associations of phospholipid-binding proteins fodrin, protein kinase C and annexin II in cultured MDCK cells.

Annexin II, alpha-fodrin and protein kinase C (PKC) are associated with the cytoplasmic surface of the plasma membranes. When assayed with liposomes, they show affinity for acidic phospholipids and bind calcium ions. They also respond to or participate in cell signal transduction by altered membrane binding properties. In the present work we have studied the properties of these proteins in epithelial MDCK cells in response to elevated intracellular calcium ion concentration, lowered pH, treatment with tumor promoter phorbol myristoyl acetate (PMA) and calmodulin inhibitor trifluoperazine (TFP). In untreated polarized MDCK cells annexin II was seen both along the lateral walls and membranes of intracellular vesicles, fodrin was located along the lateral walls, whereas PKC was seen in the cytoplasm. There was no observable translocation of these proteins upon elevation of the intracellular calcium concentration using a calcium ionophore A23187. On the other hand, treatment with TFP led to a release of annexin II from the plasma membranes which was accompanied by a transient peak in the intracellular calcium. Treatment with PMA led to a loss of the cubic form of the cells, a slight elevation in the intracellular calcium concentration and a drop in the intracellular pH. Simultaneously fodrin was released from the lateral walls, but still remained insoluble in Triton X-100, PKC became associated with the intracellular membranes and fibers, whereas annexin II remained along the lateral walls. These changes could be prevented by clamping the intracellular pH neutral during PMA treatment. On the other hand, lowering of intracellular pH below 6.5 with the nigericin treatment led to a similar translocation of fodrin and PKC as PMA. This suggests that the protein redistribution is caused by cytoplasmic acidification and is due to an increased hydrophobicity and enhanced protonation of lipids and proteins. In contrast, no changes were seen in the annexin II distribution in response to altered pH. Hence, its release by TFP is presumably due to changes in the cationic properties of the inner phase of the plasma membrane. Thus, proteins which show similar binding properties with liposomes show different characteristics in their association with the intracellular membranes.