Downregulation of rRNA transcription triggers cell differentiation

Abstract

Responding to various stimuli is indispensable for the maintenance of homeostasis. The downregulation of ribosomal RNA (rRNA) transcription is one of the mechanisms involved in the response to stimuli by various cellular processes, such as cell cycle arrest and apoptosis. Cell differentiation is caused by intra- and extracellular stimuli and is associated with the downregulation of rRNA transcription as well as reduced cell growth. The downregulation of rRNA transcription during differentiation is considered to contribute to reduced cell growth. However, the downregulation of rRNA transcription can induce various cellular processes; therefore, it may positively regulate cell differentiation. To test this possibility, we specifically downregulated rRNA transcription using actinomycin D or a siRNA for Pol I-specific transcription factor IA (TIF-IA) in HL-60 and THP-1 cells, both of which have differentiation potential. The inhibition of rRNA transcription induced cell differentiation in both cell lines, which was demonstrated by the expression of the common differentiation marker CD11b. Furthermore, TIF-IA knockdown in an ex vivo culture of mouse hematopoietic stem cells increased the percentage of myeloid cells and reduced the percentage of immature cells. We also evaluated whether differentiation was induced via the inhibition of cell cycle progression because rRNA transcription is tightly coupled to cell growth. We found that cell cycle arrest without affecting rRNA transcription did not induce differentiation. To the best of our knowledge, our results demonstrate the first time that the downregulation of rRNA levels could be a trigger for the induction of differentiation in mammalian cells. Furthermore, this phenomenon was not simply a reflection of cell cycle arrest. Our results provide a novel insight into the relationship between rRNA transcription and cell differentiation.

Figure 1. Suppression of rRNA transcription by actinomycin D (Act D) induced the differentiation of HL-60 and THP-1 cells.

(A) A low concentration of Act D inhibited rRNA transcription in HL-60 and THP-1 cells. HL-60 and THP-1 cells were treated with 5 nM Act D for 24 h. The levels of pre-rRNA were determined by real-time quantitative PCR (RT-qPCR) and normalized by cell number. (B) Act D induced the expression of CD11b in HL-60 and THP-1 cells. Cells were cultured in the absence (control) or presence of all-trans-retinoic acid (ATRA) (1 µM), PMA (10 ng/mL), or Act D (5 nM) at 37°C. After 3 days, the CD11b expression levels were determined by flow cytometry (left panels). The corresponding mean percentages of CD11b-positive cells are shown in the left panels (right panels). (C, D) Inhibition of the cell cycle did not affect CD11b expression. THP-1 cells were treated with PMA (10 ng/mL), Act D (5 nM), or roscovitine (15 µM) for 3 days. (C) The DNA content was determined by DAPI and analyzed by flow cytometry. Similar results were obtained in three independent experiments. (D) The CD 11b expression levels were determined by flow cytometry. The corresponding mean percentages of CD11b-positive cells are shown in the left panels (right panels). (E) Roscovitine treatment did not affect the pre-rRNA levels. THP-1 cells were treated with PMA (10 ng/mL), Act D (5 nM), or roscovitine (15 µM) for 3 days. The pre-rRNA levels were determined by RT-qPCR and normalized by cell number. Values are expressed as the mean ± S.D., n = 3. *P<0.05. N.S.: P>0.05.

Figure 2. Suppression of rRNA transcription by TIF-IA KD induced the differentiation of HL-60.

(A, B) siRNA-TIF-IA reduced the mRNA and protein levels of TIF-IA. HL-60 cells were transfected with siRNAs for luciferase (siCont.) and TIF-IA (siTIF-IA#1 or siTIF-IA#2), and cultured for 3 days. (A) The mRNA levels of TIF-IA were determined by RT-qPCR. (B) The protein levels of TIF-IA were determined by immunoblotting. (C) The pre-rRNA levels were determined by RT-qPCR. (D, E) TIF-IA KD induced the differentiation of HL-60 cells. (D) CD11b expression was determined by flow cytometry. ATRA (1 µM) was used as the positive control. The corresponding mean percentages of CD11b-positive cells are shown in the left panels (right panels). We present the same histograms for siCont. and ATRA in the upper and lower panels because these experiments were performed at the same time. (E) The MPO levels were determined by RT-qPCR and normalized by the cyclophilin levels. Values are expressed as the mean ± S.D., n = 3 (A, B, D). Values are expressed as the mean ± S.D., n = 4 (C). *P<0.05.

Figure 3. rRNA transcription levels decreased during mouse hematopoietic differentiation.

(A) This scheme illustrates mouse hematopoietic differentiation into neutrophils, monocytes/macrophages, erythroblasts, or megakaryocyte cells. Nine types of cells were classified as stem cell, progenitor cells, and differentiated cells. The multipotent progenitor is denoted as MPP, the common myeloid progenitor as CMP, the granulocyte-monocyte progenitor as GMP, and the megakaryocyte-erythrocyte progenitor as MEP. (B) The pre-rRNA levels were reduced in the differentiated cells. The pre-rRNA levels of the nine types of hematopoietic cells were determined by RT-qPCR and normalized by cell number. (C) The nucleolar size was reduced in the differentiated cells. The nucleoli of the nine types of hematopoietic cells were immunostained using nucleophosmin (NPM) antibodies (panels with odd numbers). Merged images with DAPI are also shown (NPM, green; DAPI, blue; panels with even numbers). The white bar represents 2 µm. Values are expressed as the mean ± S.D., n = 5.

Figure 4. TIF-IA KD-induced cell differentiation of mouse HSCs in ex vivo culture.

(A) Scheme showing the experimental procedure used for the HSC ex vivo culture system. The HSCs were purified from 8- to 12-week-old wild type mice. The purified HSCs were transduced with a lentivirus that expressed a shRNA against TIF-IA and cultured in media containing SCF and TPO. On days 5, 7, 10, and 12 after lentivirus transduction, myeloid differentiation was analyzed by flow cytometry. (B, C) TIF-IA KD promoted the myeloid differentiation of HSCs in culture. Anti-Mac-1 and anti-Gr-1 were used as myeloid cell markers. (B) The upper and lower panels show the results for the shControl and shTIF-IA cultures, respectively. Each panel shows the flow cytometric profiles of GFP⁺ transduced cells. (C) The percentages of lineage⁻ cells (Mac-1⁻Gr-1⁻) and myeloid cells (Mac1⁺ single positive and Mac-1⁺Gr-1⁺) among the GFP⁺ cells are shown as bar graphs. Values are expressed as the mean ± S.D., n = 3. *P<0.05.