Lack of cyclin D3 induces skeletal muscle fiber-type shifting, increased endurance performance and hypermetabolism.

The mitogen-induced D-type cyclins (D1, D2 and D3) are regulatory subunits of the cyclin-dependent kinases CDK4 and CDK6 that drive progression through the G1 phase of the cell cycle. In skeletal muscle, cyclin D3 plays a unique function in controlling the proliferation/differentiation balance of myogenic progenitor cells. Here, we show that cyclin D3 also performs a novel function, regulating muscle fiber type-specific gene expression. Mice lacking cyclin D3 display an increased number of myofibers with higher oxidative capacity in fast-twitch muscle groups, primarily composed of myofibers that utilize glycolytic metabolism. The remodeling of myofibers toward a slower, more oxidative phenotype is accompanied by enhanced running endurance and increased energy expenditure and fatty acid oxidation. In addition, gene expression profiling of cyclin D3-/- muscle reveals the upregulation of genes encoding proteins involved in the regulation of contractile function and metabolic markers specifically expressed in slow-twitch and fast-oxidative myofibers, many of which are targets of MEF2 and/or NFAT transcription factors. Furthermore, cyclin D3 can repress the calcineurin- or MEF2-dependent activation of a slow fiber-specific promoter in cultured muscle cells. These data suggest that cyclin D3 regulates muscle fiber type phenotype, and consequently whole body metabolism, by antagonizing the activity of MEF2 and/or NFAT.

Cyclin D3 is selectively required for proliferative expansion of germinal center B cells.

The generation of robust T-cell-dependent humoral immune responses requires the formation and expansion of germinal center structures within the follicular regions of the secondary lymphoid tissues. B-cell proliferation in the germinal center drives ongoing antigen-dependent selection and the generation of high-affinity class-switched plasma and memory B cells. However, the mechanisms regulating B-cell proliferation within this microenvironment are largely unknown. Here, we report that cyclin D3 is uniquely required for germinal center progression. Ccnd3(-/-) mice exhibit a B-cell-intrinsic defect in germinal center maturation and fail to generate an affinity-matured IgG response. We determined that the defect resulted from failed proliferative expansion of GL7(+) IgD(-) PNA(+) B cells. Mechanistically, sustained expression of cyclin D3 was found to be regulated at the level of protein stability and controlled by glycogen synthase kinase 3 in a cyclic AMP-protein kinase A-dependent manner. The specific defect in proliferative expansion of GL7(+) IgD(-) PNA(+) B cells in Ccnd3(-/-) mice defines an underappreciated step in germinal center progression and solidifies a role for cyclin D3 in the immune response, and as a potential therapeutic target for germinal center-derived B-cell malignancies.

Requirement for cyclin D3 in germinal center formation and function.

Germinal centers (GC) of secondary lymphoid tissues are critical to mounting a high-affinity humoral immune response. B cells within the GC undergo rapid clonal expansion and selection while diversifying their antibody genes. Although it is generally believed that GC B cells employ a unique proliferative program to accommodate these processes, little is known about how the GC-associated cell cycle is orchestrated. The D-type cyclins constitute an important component of the cell cycle engine that enables the cells to respond to physiological changes. Cell type- and developmental stage-specific roles of D-type cyclins have been described but the cyclin D requirement during GC reaction has not been addressed. In this study, we report that cyclin D3 is largely dispensable for proliferation and Ig class switching of in vitro activated B cells. In contrast, GC development in Ccnd3(-/-) mice is markedly impaired, as is the T cell-dependent antibody response. Within the GC, although both switched and unswitched B cells are affected by cyclin D3 inactivation, the IgM(-) pool is more severely reduced. Interestingly, despite a compensatory increase in cyclin D2 expression, a significant number of Ccnd3(-/-) GC B cells accumulate in quiescent G0 state. Lastly, although cyclin D3 inactivation did not disrupt BCL6 expression in GC B cells, it completely blocked the GC promoting effect of BCL6 overexpression, suggesting that cyclin D3 acts downstream of BCL6 to regulate GC formation. This is the first demonstration that cyclin D3 plays an important and unique role at the GC stage of B cell development.