UV radiation regulates Mi-2 through protein translation and stability

Abstract

Dermatomyositis (DM) is an autoimmune disease, which is often accompanied by the development of disease-specific autoantibodies directed against the SNF2-superfamily helicase, Mi-2. Recent evidence suggests that ultraviolet radiation exposure may be an important risk factor for the development of not only the disease but also specific autoimmunity against Mi-2. Consequently, we investigated the effects of ultraviolet radiation on Mi-2 protein expression. We observed an increase in protein levels upon ultraviolet radiation exposure in cell culture systems. These changes in expression occur quite rapidly, are maximized just 1 h following exposure, and are unique to Mi-2 when compared with other members of the NuRD complex. Changes in protein levels are not mediated through transcriptional mechanisms. Treatment results in a more efficiently translated message through regulatory elements in the 5'-UTR region of the transcript. Investigation into protein half-life further demonstrated increased stability of Mi-2 following UV exposure. Taken together, we describe a system by which Mi-2 protein expression can be quickly increased following UV exposure and then maintained up to 16 h later. These data provide a novel regulation of an important transcriptional regulator and provide insight into the possible mechanisms of the development of DM and associated autoantibodies.

FIGURE 1.

UV radiation increases Mi-2 expression. Immortalized human keratinocyte cell lines, 1106 (A) and 1102 (B), were treated with the indicated doses of UV radiation. 8 h after treatment, cells lysates were harvested, subjected to SDS-PAGE, and immunoblotted with antibodies specific for the indicated proteins. C, MCF7 cells were treated with the indicated doses of UV radiation. 8 h after treatment, cell lysates were analyzed for changes of Mi-2 protein expression. D, the 1106 keratinocyte cell line was treated with the indicated amounts of ionizing radiation. 8 h following treatment, cell lysates were harvested, and Mi-2 and β-actin protein expression were analyzed by immunoblot. E, the 1106 keratinocyte cell line was treated with 90 J/m² of UV radiation and cell lysates harvested at indicated time points. Samples were analyzed for Mi-2 and β-actin expression by immunoblot.

FIGURE 2.

UV radiation induces an S-phase checkpoint following Mi-2 induction. A, keratinocytes were treated with 90 J/m2 of UV radiation, and cells were harvested at the indicated time points for prodium iodide flow cytometry analysis. B, keratinocytes were plated onto coverslips and either mock-treated or irradiated with 90 J/m² UV. Cells were fixed at the indicated time points following UV exposure and immunocytochemistry performed to detect CPDs (green) and Mi-2 (red).

FIGURE 3.

Mi-2 induction is regulated through transcriptional mechanisms. A, keratinocytes were treated with 90 J/m² UV radiation and RNA harvested at the indicated time points. Relative mRNA for the indicated targets was analyzed by quantitative real-time RT-PCR. Data presented represents the average of six individual experiments with the S.E. ^* represents p < 0.05.

FIGURE 4.

Mi-2 is stabilized following UV radiation. A, keratinocytes were treated with cycloheximide (50 ng/ml) and 90 J/m² UV radiation as indicated. Cell lysates were harvested at the specified time points and protein expression analyzed by immunoblot. B, keratinocytes were treated with MG132 and 90 J/m2 UV radiation as indicated. 4 h after treatment, cell lysates were harvested and protein expression analyzed by immunoblot.

FIGURE 5.

Mi-2 protein translation is regulated through the 5′-UTR following UV radiation. A, the 5′- and 3′-UTRs of Mi-2β were cloned into the pMIR-Report luciferase vector as depicted. B, keratinocytes were transfected with CMV-Renilla luciferase and the indicated reporter plasmid. 24 h following transfection, cells were treated with 90 J/m² UV radiation. Cell lysates were collected 2 h following treatment and analyzed for luciferase activity. Data represent the average of at least nine independent points with S.E. C, mRNA was harvested from cells transfected and treated as in B and used for real-time RT-PCR analysis. Relative luciferase message levels are graphed corrected against Renilla luciferase message. D, MCF7 cells were mock-treated or treated with 90 J/m2 UV followed by incubation with [³⁵S]methionine containing media. 2 h after treatment, cell lysates were collected and immunoprecipitated with Mi-2 specific antibodies or purified nonspecific rabbit IgG. Precipitated proteins were subjected to SDS-PAGE and visualized by phosphorimaging. The arrows mark the Mi-2 protein and a nonspecific protein used as a loading control.