Linkage of M-CSF signaling to Mitf, TFE3, and the osteoclast defect in Mitf(mi/mi) mice.

Osteoclasts are multinucleated hematopoietic cells essential for bone resorption. Macrophage colony-stimulating factor (M-CSF) is critical for osteoclast development and function, although its nuclear targets in osteoclasts are largely unknown. Mitf and TFE3 are two closely related helix-loop-helix (HLH) transcription factors previously implicated in osteoclast development and function. We demonstrate that cultured Mitf(mi/mi) osteoclasts are immature, mononuclear, express low levels of TRAP, and fail to mature upon M-CSF stimulation. In addition, M-CSF induces phosphorylation of Mitf and TFE3 via a conserved MAPK consensus site, thereby triggering their recruitment of the coactivator p300. Furthermore, an unphosphorylatable mutant at the MAPK consensus serine is specifically deficient in formation of multinucleated osteoclasts, mimicking the defect in Mitf(mi/mi) mice. These results identify a signaling pathway that appears to coordinate cytokine signaling with the expression of genes vital to osteoclast development.

Contingent neutrality in competing viral populations.

The replicative fitness of a genetically marked (MARM-C) population of vesicular stomatitis virus was examined in competition assays in BHK-21 cells. In standard fitness assays involving up to eight competition passages of the mixed populations, MARM-C competes equally with the wild type (wt), but very prolonged competitions always led to the wt gaining dominance over MARM-C in a very slowed, nonlinear manner (J. Quer et al., J. Mol. Biol. 264:465-471, 1996). In the present study we show that a number of quite unrelated environmental perturbations, which decreased virus replication during competitions, all led to an accelerated dominance of the wt over MARM-C. These perturbations were (i) the presence of added (or endogenously generated) defective interfering particles, (ii) the presence of the chemical mutagen 5-fluorouracil (5-FU), or (iii) an increase in temperature to 40.5 degrees C. Thus, the "neutral fitness" of the MARM-C population is contingent. We have determined the entire genomic consensus sequence of MARM-C and have identified only six mutations. Clearly, some or all of these mutations allowed the MARM-C quasispecies population to compete equally with wt in a defined constant host environment, but the period of neutrality was shortened when the environment was perturbed during competitions. Interestingly, when four passages of each population were carried out independently in the presence of 5-FU (but in the absence of competition), no significant differences were detected in the fitness changes of wt and MARM-C, nor was there a difference in their subsequent abilities to compete with each other in a standard fitness assay. We propose a model for this contingent neutrality. The conditions employed to generate the MARM-C quasispecies population selected a small number of mutations in the consensus sequence. It appears that the MARM-C quasispecies population has moved into a segment of sequence space in which the average fitness value is neutral but, under environmental stress, beneficial mutations cannot be generated rapidly enough to compete with those being generated concurrently by competing wt virus quasispecies populations.

Lack of evolutionary stasis during alternating replication of an arbovirus in insect and mammalian cells.

The evolution of vesicular stomatitis virus (VSV) in a constant environment, consisting of either mammalian or insect cells, has been compared to the evolution of the same viral population in changing environments consisting in alternating passages in mammalian and insect cells. Fitness increases were observed in all cases. An initial fitness loss of VSV passaged in insect cells was noted when fitness was measured in BHK-21 cells, but this effect could be attributed to a difference of temperature during VSV replication at 37 degrees C in BHK-21 cells. Sequencing of nucleotides 1-4717 at the 3' end of the VSV genome (N, P, M and G genes) showed that at passage 80 the number of mutations accumulated during alternated passages (seven mutations) is similar or larger than that observed in populations evolving in a constant environment (two to four mutations). Our results indicate that insect and mammalian cells can constitute similar environments for viral replication. Thus, the slow rates of evolution observed in natural populations of arboviruses are not necessarily due to the need for the virus to compromise between adaptation to both arthropod and vertebrate cell types.

Age-resolving osteopetrosis: a rat model implicating microphthalmia and the related transcription factor TFE3.

Microphthalmia (Mi) is a basic helix-loop-helix-leucine zipper (b-HLH-ZIP) transcription factor implicated in pigmentation, mast cells, and bone development. Two dominant-negative mi alleles (mi/mi and Mior/Mior) in mice cause osteopetrosis. In contrast, osteopetrosis has not been observed in a number of recessive mi alleles, suggesting the existence of Mi protein partners important in osteoclast function. An osteopetrotic rat of unknown genetic defect (mib) has been described whose skeletal sclerosis improves dramatically with age and that is associated with pigmentation defects reminiscent of mouse mi alleles. Here we report that this rat strain harbors a large genomic deletion encompassing the 3' half of mi including most of the b-HLH-ZIP region. Osteoclasts from these animals lack Mi protein in contrast to wild-type rat, mouse, and human osteoclasts. Mi is not detectable in primary osteoblasts. In addition TFE3, a b-HLH-ZIP transcription factor related to Mi, was found to be expressed in osteoclasts, but not osteoblasts, and to coimmunoprecipitate with Mi. These results demonstrate the existence of members of a family of biochemically related transcription factors that may cooperate to play a central role in osteoclast function and possibly in age-related osteoclast homeostasis.