RanBPM has proapoptotic activities that regulate cell death pathways in response to DNA damage.

Ran-binding protein M (RanBPM) is a nucleocytoplasmic protein previously implicated in various signaling pathways, but whose function remains enigmatic. Here, we provide evidence that RanBPM functions as an activator of apoptotic pathways induced by DNA damage. First, transient expression of RanBPM in HeLa cells induced cell death through caspase activation, and in the long-term, forced expression of RanBPM impaired cell viability. RanBPM COOH-terminal domain stimulated the ability of RanBPM to induce caspase activation, whereas this activity was negatively regulated by the central SPRY domain. Second, small interfering RNA-directed knockdown of RanBPM prevented DNA damage-induced apoptosis, as evidenced by the marked reduction in caspase-3 and caspase-2 activation. This correlated with a magnitude fold increase in the survival of RanBPM-depleted cells. Following ionizing radiation treatment, we observed a progressive relocalization of RanBPM from the nucleus to the cytoplasm, suggesting that the activation of apoptotic pathways by RanBPM in response to ionizing radiation may be regulated by nucleocytoplasmic trafficking. Finally, RanBPM downregulation was associated with a marked decrease of mitochondria-associated Bax, whereas Bcl-2 overall levels were dramatically upregulated. Overall, our results reveal a novel proapoptotic function for RanBPM in DNA damage-induced apoptosis through the regulation of factors involved in the mitochondrial apoptotic pathway.

The DP-1 transcription factor is required for keratinocyte growth and epidermal stratification.

The epidermis is a stratified epithelium constantly replenished through the ability of keratinocytes in its basal layer to proliferate and self-renew. The epidermis arises from a single-cell layer ectoderm during embryogenesis. Large proliferative capacity is central to ectodermal cell and basal keratinocyte function. DP-1, a heterodimeric partner of E2F transcription factors, is highly expressed in the ectoderm and all epidermal layers during embryogenesis. To investigate the role of DP-1 in epidermal morphogenesis, we inhibited DP-1 activity through exogenous expression of a dominant-negative mutant (dnDP-1). Expression of the dnDP-1 mutant interferes with binding of E2F/DP-1 heterodimers to DNA and inhibits DNA replication, as well as cyclin A mRNA and protein expression. Chromatin immunoprecipitation analysis demonstrated that the cyclin A promoter is predominantly bound in proliferating keratinocytes by complexes containing E2F-3 and E2F-4. Thus, the mechanisms of decreased expression of cyclin A in the presence of dnDP-1 seem to involve inactivation of DP-1 complexes containing E2F-3 and E2F-4. To assess the consequences on epidermal morphogenesis of inhibiting DP-1 activity, we expressed dnDP-1 in rat epithelial keratinocytes in organotypic culture and observed that DP-1 inhibition negatively affected stratification of these cells. Likewise, expression of dnDP-1 in embryonic ectoderm explants produced extensive disorganization of subsequently formed epidermal basal and suprabasal layers, interfering with normal epidermal formation. We conclude that DP-1 activity is required for normal epidermal morphogenesis and ectoderm-to-epidermis transition.