Increased osteoclastic resorption and subsequent bone loss are common features of many debilitating diseases including osteoporosis, bone metastases, Paget's disease, and rheumatoid arthritis. While rapid progress has been made in elucidating the signaling pathways directing osteoclast differentiation and function, a comprehensive picture is far from complete. Here, we explore the role of the Ca 2þ -activated regulator calmodulin in osteoclastic differentiation, functional bone resorption, and apoptosis. During active bone resorption, calmodulin expression is increased, and calmodulin concentrates at the ruffled border, the organelle utilized for acid transport and bone dissolution. Pharmacologic inhibitors of calmodulin, several of which are already used clinically as anti-cancer and anti-psychotic agents, inhibit osteoclastic acid transport, suggesting their potential as bone-sparing drugs. Recent studies also implicate calmodulin in osteoclast apoptosis through a mechanism involving its direct interaction with the death receptor Fas. During osteoclastogenesis, RANKL-induction stimulates a rise in intracellular Ca 2þ , which in turn activates calmodulin and its downstream effectors. In particular, the Ca 2þ /calmodulin-dependent phosphatase calcineurin and its targets, the NFAT family of transcription factors, have been posited as the master regulators of osteoclastogenesis. However, recent in vivo and in vitro studies demonstrate that another Ca 2þ /calmodulin-regulated effector protein, CaMKII, is also involved. CaMKII þ/À mutant mice have reduced osteoclast numbers, and CaMKII antagonists inhibit osteoclastogenesis in vitro. Furthermore, CaMKII is known to activate AP-1 transcription factors, which are also required for RANKL-induced osteoclast gene transcription, and recent findings suggest that CaMKII can down-regulate gp130, a cytokine receptor involved in bone remodeling and implicated in numerous osteo-articular diseases.

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We and others have demonstrated that Fas-mediated apoptosis is a potential therapeutic target for cholangiocarcinoma. Previously, we reported that CaM (calmodulin) antagonists induced apoptosis in cholangiocarcinoma cells through Fas-related mechanisms. Further, we identified a direct interaction between CaM and Fas with recruitment of CaM into the Fas-mediated DISC (death-inducing signalling complex), suggesting a novel role for CaM in Fas signalling. Therefore we characterized the interaction of CaM with proteins recruited into the Fas-mediated DISC, including FADD (Fas-associated death domain)-containing protein, caspase 8 and c-FLIP {cellular FLICE [FADD (Fas-associated death domain)-like interleukin 1beta-converting enzyme]-like inhibitory protein}. A Ca(2+)-dependent direct interaction between CaM and FLIP(L), but not FADD or caspase 8, was demonstrated. Furthermore, a 37.3+/-5.7% increase (n=6, P=0.001) in CaM-FLIP binding was observed at 30 min after Fas stimulation, which returned to the baseline after 60 min and correlated with a Fas-induced increase in intracellular Ca(2+) that reached a peak at 30 min and decreased gradually over 60 min in cholangiocarcinoma cells. A CaM antagonist, TFP (trifluoperazine), inhibited the Fas-induced increase in CaM-FLIP binding concurrent with inhibition of ERK (extracellular-signal-regulated kinase) phosphorylation, a downstream signal of FLIP. Direct binding between CaM and FLIP(L) was demonstrated using recombinant proteins, and a CaM-binding region was identified in amino acids 197-213 of FLIP(L). Compared with overexpression of wild-type FLIP(L) that resulted in decreased spontaneous as well as Fas-induced apoptosis, mutant FLIP(L) with deletion of the CaM-binding region resulted in increased spontaneous and Fas-induced apoptosis in cholangiocarcinoma cells. Understanding the biology of CaM-FLIP binding may provide new therapeutic targets for cholangiocarcinoma and possibly other cancers.

One hallmark of AIDS progression is a decline in CD4+ T lymphocytes, though the mechanism is poorly defined. There is ample evidence that increased apoptosis is responsible for some, if not all, of the decline. Prior studies have shown that binding of cellular calmodulin to the envelope glycoprotein (Env) of HIV-1 increases sensitivity to fas-mediated apoptosis and that calmodulin antagonists can block this effect. We show that individual mutation of five residues in the C-terminal calmodulin-binding domain of Env is sufficient to significantly reduce fas-mediated apoptosis in transfected cells. The A835W mutation in the cytoplasmic domain of gp41 eliminated co-immunoprecipitation of Env with calmodulin in studies with stably transfected cells. Four point mutations (A835W, A838W, A838I, and I842R) and the corresponding region of HIV-1 HXB2 were cloned into the HIV-1 proviral vector pNL4-3 with no significant effect on viral production or envelope expression, although co-immunoprecipitation of calmodulin and Env was decreased in three of these mutant viruses. Only wild-type envelope-containing virus induced significantly elevated levels of spontaneous apoptosis by day 5 post-infection. Fas-mediated apoptosis levels positively correlated with the degree of calmodulin co-immunoprecipitation, with the lowest apoptosis levels occurring in cells infected with the A835W envelope mutation. While spontaneous apoptosis appears to be at least partially calmodulin-independent, the effects of HIV-1 Env on fas-mediated apoptosis are directly related to calmodulin binding.

The envelope (Env) glycoprotein of Mason-Pfizer monkey virus (M-PMV), like those of other retroviruses, is synthesized on the rough endoplasmic reticulum (ER) and is cotranslationally glycosylated and inserted into the lumen of the ER (5-8, 30). Shortly after synthesis, the glycosylated precursor is assembled into trimers, a process which is thought to be required for transport of Env from the ER to the Golgi complex (2,20). It is then cleaved by a cellular protease into two subunits, gp70 (SU) and gp22 (TM), in a late compartment of the Golgi complex (26). The oligomeric, noncovalently associated gp70 and gp22 complexes are then transported to the plasma membrane, where they are incorporated into budding virions (10, 68). The SU glycoprotein is responsible for receptor binding, whereas the TM glycoprotein is responsible for anchoring the SU protein at the surface of infected cells or the viral membrane. The TM glycoprotein also mediates virus-cell membrane fusion during viral entry as well as cell-cell fusion via a fusion peptide and heptad repeat motifs located at the extracellular domain (2,18,35,69,74,76). This fusion process also is influenced by the cytoplasmic domain of the TM glycoprotein as demonstrated previously (9,13,19,36,44,55,68,70).As is observed with murine leukemia virus (MuLV) and Gibbon ape leukemia virus, but unlike most other retroviruses, a viral protease-mediated maturational cleavage of the TM cytoplasmic domain occurs following virus release, which results in conversion of gp22 into gp20 (9,10,13,55,67). Based on cytoplasmic domain truncation mutants, this maturational cleavage of the cytoplasmic domain appears to dramatically increase the fusion activity of the TM proteins and results in the loss of 17 amino acids from the carboxy terminus of the cytoplasmic domain (9, 68).The incorporation of glycoprotein into budding virions is essential for the formation of an infectious virus particle, since retrovirus Env proteins play important roles in receptor binding and membrane fusion. In the case of the alphaviruses, an interaction between the cytoplasmic domain of the spike glycoprotein and the virus nucleocapsid has been demonstrated directly and is absolutely required for virus budding (1,23,72,87). For retroviruses, which do not require glycoprotein expression for virus assembly and release, the nature of capsidenvelope interactions is less well defined. In M-PMV, the glycoprotein appears to play an important role in intracellular transport of assembled capsids to the plasma membrane, and mutations that interfere with Env incorporation also decrease the efficiency of virus release (64,65,68). In contrast, Rous sarcoma virus, which encodes a glycoprotein lacking a cytoplasmic domain, can efficiently assemble and infect cells (53). In the case of Moloney MuLV, some deletion mutations in the cytoplasmic domain of the TM protein decrease infectivity without reducing glycoprotein incorporation (33). Evidence derived from Env and Gag mutagenesis and pseudotyping