Cold-inducible RNA-binding protein (Cirp) interacts with Dyrk1b/Mirk and promotes proliferation of immature male germ cells in mice

Abstract

Cold-inducible RNA-binding protein (Cirp) was the first cold-shock protein identified in mammals. It is structurally quite different from bacterial cold-shock proteins and is induced in response to mild, but not severe, hypothermia. To clarify the physiological function of Cirp in vivo, we produced cirp-knockout mice. They showed neither gross abnormality nor defect in fertility, but the number of undifferentiated spermatogonia was significantly reduced and the recovery of spermatogenesis was delayed after treatment with a cytotoxic agent, busulfan. Cirp accelerated cell-cycle progression from G0 to G1 as well as from G1 to S phase in cultured mouse embryonic fibroblasts. Cirp directly bound to dual-specificity tyrosine-phosphorylation-regulated kinase 1B (Dyrk1b, also called Mirk) and inhibited its binding to p27, resulting in decreased phosphorylation and destabilization of p27. Cirp did not affect binding of Dyrk1b to cyclin D1 but inhibited phosphorylation of cyclin D1 by Dyrk1b, resulting in cyclin D1 stabilization. In the spermatogonial cell line GC-1spg, suppression of Cirp expression increased the protein level of p27, decreased that of cyclin D1, and decreased the growth rate, which depended on Dyrk1b. Consistent changes in the protein levels of p27 and cyclin D1 as well as the percentage of cells in G0 phase were observed in undifferentiated spermatogonia of cirp-knockout mice. In undifferentiated spermatogonia of wild-type mice, Cirp and Dyrk1b colocalized in the nucleus. Thus, our study demonstrates that Cirp functions to fine-tune the proliferation of undifferentiated spermatogonia by interacting with Dyrk1b.

Fig. 1.

Reduced number of undifferentiated spermatogonia in cirp-knockout mice. (A) Targeted disruption of the cirp gene. Structures of the wild-type allele, targeting vector, and mutant allele are shown together with the relevant restriction sites. The filled rectangles indicate the exons of the cirp gene. Neo, TK, and arrows (a and b) indicate a pgk-neo selection cassette, a thymidine kinase cassette, and the locations of primers for genomic PCR, respectively. (B) Flow cytometric analysis of undifferentiated spermatogonia. The fractions of undifferentiated spermatogonia (boxed) were compared between wild-type (+/+) and cirp-knockout (−/−) mice. *Significantly different from +/+ mice (P < 0.05). Data are from four +/+ and three −/− 12-wk-old mice. (C) Numbers of undifferentiated spermatogonia in +/+, −/−, and heterozygous knockout (+/−) mice. Significant decrease (*P < 0.05) was seen in −/− mice but not in +/− mice (P = 0.93). Values represent mean ± SD. Data are from eight +/+, three −/−, and four +/− mice. (D) Percentage of reconstituted seminiferous tubules after busulfan treatment. *Significantly different (P < 0.05). **Significantly different (P < 0.01). Values are mean ± SD (n = 3 each). (E) Histological sections of the testes at 7 wk after busulfan treatment. Sections are stained with hematoxylin and eosin. (Scale bar: 50 μm.) (F) Numbers of undifferentiated spermatogonia after busulfan treatment. ● and ○ indicate the numbers in +/+ and −/− mice, respectively. Values represent mean ± SD (n = 3–7). **Significantly different between +/+ and −/− mice (P < 0.01).

Fig. 2.

Accelerated proliferation of MEFs expressing Cirp. (A and B) Effects of Cirp on cell growth. Cells from Cirp(−/−)/teton-Cirp clones 2, 5, and 6 (Cl. 2, Cl. 5, and Cl. 6, respectively) were derived from cirp^−/− MEFs that express Cirp in the presence of Dox. Cirp(+/+)/teton-shCirp cells were derived from cirp^+/+ MEFs that Dox-inducibly express shRNAs that knockdown Cirp expression (shCirp-1 and shCirp-2) or scrambled RNAs (shSCR). Cells were cultured in the presence (+) or absence (−) of Dox at the indicated temperatures. Cell numbers were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Values are mean ± SD (n = 3 or 4). *P < 0.05. **P < 0.01. (C) Analysis of the cell cycle by flow cytometry. BrdU incorporation and DNA content were analyzed in indicated cells cultured at 32 °C in the presence (+) or absence (−) of Dox. Percentages of cells in indicated phases of cell cycle are shown. Values are mean ± SD (n = 3). *P < 0.05. **P < 0.01. (D and E) Percentages of cells in G0 phase in the presence (+) or absence (−) of Dox. Ki67 expression, stainability with pyronin Y, and DNA content were analyzed in indicated cells by flow cytometry. Values are mean ± SD (n = 3). *P < 0.05.

Fig. 3.

Inhibition of Dyrk1b kinase activity by Cirp. (A) Effects of Cirp on binding of p27 to Dyrk1b. Plasmids were cotransfected into HEK293T cells as indicated. Proteins immunoprecipitated (IP) with anti-FLAG antibody and inputs were analyzed by Western blotting (IB) using the indicated antibodies. +, Present; −, absent. (B) Effects of Cirp on in vitro kinase activity of Dyrk1b toward p27. Immunoprecipitants (IP) were prepared from HEK293T cells expressing FLAG-Dyrk1b by using anti-FLAG antibody. Their kinase activity was assayed in the presence of [γ-³²P]ATP, GST-p27, and increasing amounts of GST-Cirp or GST. Reaction mixtures were separated by SDS/PAGE and analyzed by autoradiography. Proteins coprecipitated with protein G Sepharose beads (*) from the reaction mixtures without ³²P and inputs (2%) were analyzed by IB using the indicated antibodies. (C) Effects of Cirp on in vitro kinase activity of Dyrk1b toward cyclin D1. Recombinant GST-Dyrk1b was incubated with [γ-³²P]ATP, GST-cyclin D1, and increasing amounts of His-tagged Cirp and analyzed as in B. The lower images show Coomassie blue staining (CBB) of the indicated proteins used in the assay and IB of proteins coprecipitated with glutathione Sepharose beads from the reaction mixtures without ³²P (affinity-purified). *1/4 of input. Experiments were repeated three (A) and four (B and C) times, with similar results.

Fig. 4.

Effects of Cirp on p27 and cyclin D1 via Dyrk1b in cultured MEFs. (A) Effects of Cirp on p27 and cyclin D1 protein levels. Cirp(−/−)/teton-Cirp Cl. 5 and Cirp(+/+)/teton-shCirp-1 cells were cultured at 32 °C in the presence (+) or absence (−) of Dox. Cell lysates were analyzed by IB using the indicated antibodies. (B) Dyrk1b dependency of protein levels. Indicated cells, both derived from Cirp(−/−)/teton-Cirp Cl. 5 cells, were cultured at 37 °C and analyzed as in A. (C) Dyrk1b and cell growth. Indicated cells were cultured at 32 °C and 37 °C in the presence (+) or absence (−) of Dox. Cell numbers were assessed by MTT assays. Values are mean ± SD (n = 3). *P < 0.05. **P < 0.01. Experiments were repeated four (B) and three (C) times, with similar results.

Fig. 5.

Impaired cell-cycle progression in Cirp-deficient spermatogonia. (A) Effects of Cirp on growth of cultured GC-1spg cells. Cells were transduced with retrovirus [murine stem cell virus-LTRmiR30-PIG (MSCV-LMP)] expressing RNAi sequences targeted to Cirp (Cirp-i-1 and Cirp-i-2) or scrambled sequence (Cirp-SCR). Cells were cultured at 32 °C, and cell numbers were assessed by MTT assays. wt, untransduced cells. Values are mean ± SD (n = 3). **P < 0.01. (B) Dyrk1b-dependent effects of Cirp on growth of GC-1spg cells. Cirp-i-1 cells were further transduced with retrovirus [Friend spleen focus-forming virus-based hybrid vector 2 (SF2)] expressing RNAi sequences targeted to Dyrk1b (Dyrk-i-1 and Dyrk-i-2) or scrambled sequence (Dyrk-SCR) and analyzed as in A. (C–E) Undifferentiated spermatogonia of cirp^−/− and cirp^+/+ genotypes were sorted and analyzed at 4 wk after busulfan treatment. (C) The percentages of Ki67⁺ (Left) and pyronin Y⁻ or pyronin Y^low (Right) cells were assessed by flow cytometry. Values are mean ± SD [n = 3 (Left) or 4 (Right)]. *P < 0.05 and **P < 0.01, respectively, different between wild-type (+/+) and cirp-knockout (−/−) mice. (D) Detection of p27 and cyclin D1 transcripts in spermatogonia isolated from cirp^−/− mice. cDNAs were analyzed by RT-PCR. (E) Protein levels of p27 and cyclin D1. Cell lysates were analyzed by IB using the indicated antibodies. Experiments were repeated three times (D and E), with similar results.