Selection of a Dominant Negative Retinoblastoma Protein (RB) Inhibiting Satellite Myoblast Differentiation Implies an Indirect Interaction between MyoD and RB

Abstract

Satellite myoblasts serve as stem cells in postnatal skeletal muscle, but the genes responsible for choosing between growth versus differentiation are largely undefined. We have used a novel genetic approach to identify genes encoding proteins whose dominant negative inhibition is capable of interrupting the in vitro differentiation of C2C12 murine satellite myoblasts. The screen is based on fusion of a library of cDNA fragments with the lysosomal protease cathepsin B (CB), such that the fusion protein intracellularly diverts interacting factors to the lysosome. Among other gene fragments selected in this screen, including those of known and novel sequence, is the retinoblastoma protein (RB) pocket domain. This unique dominant negative form of RB allows us to genetically determine if MyoD and RB associate in vivo. The dominant negative CB-RB fusion produces a cellular phenotype indistinguishable from recessive loss of function RB mutations. The fact that the dominant negative RB inhibits myogenic differentiation in the presence of nonlimiting concentrations of either RB or MyoD suggests that these two proteins do not directly interact. We further show that the dominant negative RB inhibits E2F1 but cannot inhibit a forced E2F1-RB dimer. Therefore, E2F1 is a potential mediator of the dominant negative inhibition of MyoD by CB-RB during satellite cell differentiation. We propose this approach to be generally suited to the investigation of gene function, even when little is known about the pathway being studied.Satellite cells are a lineage derived from somites that reside under the basement membrane of the myofiber and are responsible for replenishing skeletal muscle during growth in the postnatal period and in response to exercise and injury in the adult animal (24). As muscles hypertrophy, the satellite cells divide and fuse in order to increase the complement of myonuclei in myofibers. Transplant studies in chick embryos indicate that satellite cells are unable to take part in muscle embryogenesis, suggesting that they serve a specialized function as a presumptive skeletal muscle stem cell (3). Satellite cells are required to both proliferate and terminally differentiate, but invariably the proportion of satellite cells in a muscle remains constant, independent of both the age of the animal and the size of the muscle, and returns to this fixed value at the conclusion of muscle regeneration following injury (23).Understanding the molecular mechanisms responsible for the switch between proliferation and differentiation in satellite cells could be a key to understanding skeletal muscle regeneration in response to disease and trauma. However, relatively little is known about the mechanisms at work to ensure maintenance of the satellite cell population. Most of this knowledge comes from embryological studies of mesoderm differentiation in somites or the developing limb bud that have been extrapolated to tissue culture models of satellite myoblast differentiation. As with embryonic myogenesis, expression of MyoD or t...

Targeted Disruption of the Murine Bin1/Amphiphysin II Gene Does Not Disable Endocytosis but Results in Embryonic Cardiomyopathy with Aberrant Myofibril Formation

“…Five other splice isoforms [Bin1(ϩ10) and at least four Bin1(Ϫ10 ϩ 12) isoforms] exhibit tissue-specific expression. Expression of Bin1(ϩ10), the isoform originally identified in the Myc two-hybrid screen, is restricted primarily to skeletal muscle, and this isoform has been implicated in mouse myoblast differentiation (31,34,61). Further supporting a role in muscle cells, Bin1(ϩ10) has been localized to T tubules (7), and recent studies involving disruption of the likely Bin1 ortholog in Drosophila suggest that it functions in T-tubule organization (45).…”

The retinoblastoma gene is involved in multiple aspects of stem cell biology

“…By selection from the transfected cells of the clones capable of cell cycle re-entry and continued growth following serum starvation the library was enriched for cDNA molecules whose dominant-negative inhibition impair myoblast terminal differentiation. Among the several genes selected with this screen they identify the RB gene demonstrating a fundamental role in stem cell specification and differentiation (Li et al, 2000).…”

“…Other studies demonstrated that RB has a role also during early phases of myogenesis in satellite myoblasts that are the reservoir of stem cells in postnatal skeletal muscles. Li et al (2000) investigated the pathways responsible for choice between differentiation and growth in satellite stem cells by ectopically expressing dominant-negative proteins that were capable of impairing muscle differentiation. To this end, they fused a library of cDNAs from skeletal muscles with lysosomal protease cathepsin B.…”

hob1+, the fission yeast homolog of Bin1, is dispensable for endocytosis or actin organization, but required for the response to starvation or genotoxic stress

“…Neuronal Bin1 isoforms, suggested to participate in endocytosis like amphiphysin, include alternately spliced regions encoded by exons 12A-D, which mediate interactions with clathrin and other endocytotic proteins (David et al, 1996;Ramjaun and McPherson, 1998). These isoforms are functionally distinct from the ubiquitous or muscle-specific isoforms of Bin1, which localize to the nucleus as well as the cytosol, bind to the nuclear proteins c-Myc and/or c-Abl, and exhibit tumor suppressor, prodifferentiation and proapoptotic properties (Sakamuro et al, 1996;Wechsler-Reya et al, 1998;Galderisi et al, 1999;Ge et al, 1999Ge et al, , 2000aMao et al, 1999;Elliott et al, 2000;Hogarty et al, 2000;Li et al, 2000;DuHadaway et al, 2001). The ubiquitous and muscle-specific isoforms of Bin1 do not affect endocytosis (Elliott et al, 2000).…”