Regulation of mRNA Stability by Heat Shock Cognate Protein 70 (Hsc70) under the Control of Cytokines.

Downstream pathways of cytokines promoting cell division and survival have long been a focus of intensive research. p27^KIP1, a cyclin-dependent kinase inhibitor, and Bim, a BH3-only cell death activator, are downregulated by cytokines and are known to be key factors in mechanisms of cytokine-induced growth and survival. In murine IL-3-dependent Baf-3 cells, Bim and p27 are regulated by IL-3 at the mRNA levels. To test whether Bim is transcriptionally regulated, we identified cis -regulating elements of Bim gene in Baf-3 cells, but none of them are under the control of IL-3. Then we tested a possibility that mRNA stability is controlled by IL-3. β-globin mRNA enforcedly expressed in Baf-3 cells were stable either in the presence or absence of IL-3. When its 3′ untranslated region (3′UTR) was replaced by that of Bim mRNA, stability of the fusion message was impaired in cells cultured in the presence of IL-3, but not in the absence of IL-3. Then we measured mRNA half-life in cytosol (S100 extract). Radiolabeled Bim mRNA with the 5′ cap and a poly(A) tail of 100 residues synthesized in vitro was stable in cytosol from Baf-3 cells cultured in the absence of IL-3 (half-life: 30 min), but was degraded quickly in the presence of IL-3 (half-life: less than 10 min). By contrast, cap-/poly(A)- mRNA was unstable in cytosol from cells cultured with or without IL-3. The half-life of p27 mRNA was also elongated by IL-3 starvation, while the stability of c-fos mRNA was not changed. Condensation of proteins bound to mRNA using RNA-affinity chromatography revealed that a protein of around 75 KDa bound to the 3′UTR of Bim and p27 mRNA (but not c-fos mRNA) more avidly in cells cultured without IL-3 than in cells with IL-3. Mass spectrometry and immunoblot analysis identified this protein to be Hsc70. Hsc70 was reported to bind to AU-rich RNA sequences such as AUUUA avidly, but its biological significance was not known. We found that Hsc70 acts as an mRNA stabilizing factor by immunodepletion study. Because the protein expression level of Hsc70 in Baf-3 cells was not increased by IL-3 starvation, IL-3 likely inhibits RNA-binding potential of Hsc70. To obtain insights into its mechanism, we measured ATPase activity of Hsc70 protein complex. We found that the complex formed in Baf-3 cells cultured in the presence of IL-3 has high ATPase activity (i.e., Hsc70 forms ATP-bound form preferentially), while that in the absence of IL-3 is no more than background levels (i.e., Hsc70 forms ADP-bound form), raising a possibility that the binding potential to Bim mRNA and ATP/ADP-bound status of Hsc70 is related. Since cochaperones bind to Hsc70 and regulate its ATPase activity, we tested their expression and binding capacity to Hsc70. We found that IL-3 upregulates the expression of Bag-4 (also SODD) and the potential of CHIP to bind to Hsc70 (these two cochaperones are known to induce Hsc70 to ATP-bound form). Moreover, IL-3 downregulates the potential of HIP and Hsp40 to bind to Hsc70 (they are known to induce to ADP-bound form). Subsequently, Hsc70 protein complex is considered to form the ATP-bound status and has less binding ability to Bim mRNA. This novel gene-specific mechanism for the regulation of mRNA stability by the chaperone/cochaperone complex may play important roles in hematopoiesis.