PP242 | Epoxide Hydrolase

Biochemical and Biophysical Research Communications

1,25-Dihydroxyvitamin D3 induces human myeloid cell differentiation via the mTOR signaling pathway
Yongjin Kim a, Hee Suk Kim a, Jeongwon Sohn a, **, Jong Dae Ji b, *
a Department of Biochemistry, College of Medicine, Korea University, Seoul, South Korea
b Rheumatology, College of Medicine, Korea University, Seoul, South Korea

A R T I C L E I N F O

Article history:
Received 10 September 2019
Accepted 23 September 2019 Available online xxx

Keywords:
1,25-Dihydroxyviamin D3 1,25(OH)2D3
Phosphatidic acid mTOR
CEBP/b
Differentiation

Abbreviations:
1,25(OH)2D3
1,25-Dihydroxyvitamin D3 mTOR
Mammalian target of rapamycin Raptor
Regulatory-associated protein of mTOR Rictor
Rapamycin-insensitive companion of mTOR PLD
Phospholipase D siRNA
Small-interfering RNA

A B S T R A C T

1,25-Dihydroxyvitamin D3 or 1,25(OH)2D3 is known to play an important role in the differentiation of human myeloid cells. However, the molecular mechanism underlying the 1,25(OH)2D3-mediated dif- ferentiation of human myeloid cells is incompletely understood. Here, we report that 1,25(OH)2D3 in- duces differentiation of human myeloid cell lines such as U937 and THP-1 cells via the mammalian target of rapamycin (mTOR) signaling pathway. Both the expression of the differentiation marker CD14 and activation of the mTOR signaling pathway were induced by 1,25(OH)2D3 in phorbol 12-myristate 13- acetate (PMA)-differentiated U937 and THP-1 cells. The 1,25(OH)2D3-induced expression of CD14 in PMA-differentiated U937 and THP-1 cells was prevented by mTOR inhibitors, PP242 and Torin1. The 1,25(OH)2D3-induced morphological changes as characteristics of differentiated myeloid cells were also reversed after PP242 and Torin1 treatment. Silencing of either regulatory-associated protein of mTOR (Raptor) or rapamycin-insensitive companion of mTOR (Rictor) in PMA-differentiated THP-1 cells with small-interfering RNA resulted in the inhibition of CD14 expression and morphological changes induced by 1,25(OH)2D3, indicating that both mTORC1 and mTORC2 were important for the differentiation of myeloid THP-1 cells. Previous studies have shown that phosphatidic acid (PA) maintains the stability of the mTOR complex. Here we found that the attenuation of PA production with 1-butanol or a PLD in- hibitor prevented the 1,25(OH)2D3-induced upregulation of CD14. Taken together, our results show that 1,25(OH)2D3 enhances the differentiation of human myeloid cells through the mTOR signaling pathway.
© 2019 Elsevier Inc. All rights reserved.

1. Introduction

The steroid hormone 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) plays critical roles by regulating numerous cellular and physiolog- ical responses. For instance, the treatment of human myeloid cell lines such as HL-60, U937, and THP-1 with 1,25(OH)2D3 results in their differentiation into cells exhibiting functional properties and expressing differentiation markers of monocytes and/or

Corresponding author. Rheumatology, College of Medicine, Korea University, 73 Inchon-Ro, Sungbuk-Gu, Seoul, 136-705, South Korea.
Corresponding author. Department of Biochemistry, Korea University College of Medicine, Moonsook Medical Hall, 73 Inchon-Ro, Sungbuk-Gu, Seoul, 136-705, South Korea.
E-mail addresses: [email protected] (J. Sohn), [email protected] (J.D. Ji).

macrophages such as CD14 [1e3]. Despite many previous studies on 1,25(OH)2D3-induced cell differentiation, the precise molecular mechanism underlying 1,25(OH)2D3-induced differentiation is unclear.
We hypothesized that mammalian target of rapamycin (mTOR) is involved in 1,25(OH)2D3-induced myeloid cell differentiation, given its important role in the differentiation of human myeloid cell lines such as HL-60 [4,5]. mTOR, a highly conserved Ser/Thr kinase, is an evolutionarily conserved sensor of essential nutrients needed for the synthesis of biological molecules. mTOR responds to amino acids, glucose, and energy and exists in two structurally and func- tionally distinct complexes known as complex 1 (mTORC1) and complex 2 (mTORC2). mTORC1 comprises mTOR, regulatory- associated protein of mTOR (Raptor), PRAS40, Deptor, and mLST8. mTORC1 is rapamycin-sensitive and activated by growth factors,

nutrients, stress, and energy signals. Two of the major downstream targets of mTORC1 include S6K1 and 4EBP1, which are involved in the mTORC1-mediated regulation of protein synthesis and ribo- some biogenesis [6e8]. mTORC2 comprising a complex of mTOR, rapamycin-insensitive companion of mTOR (Rictor), Protor, mSIN1, Deptor, and mLST8 is rapamycin-insensitive and regulates cyto- skeletal organization, cell survival, and metabolism. mTORC2 phosphorylates AGC kinases (protein kinase A, G, and C families) such as protein kinase B (AKT), serine/threonine protein kinase 1 (SGK1), and protein kinase C (PKC) [8].
Previous reports have revealed the requirement of phosphatidic acid (PA) for the stability of both mTORC1 and mTORC2 complexes [4,5,9]. PA has many diverse roles in cell physiology. PA is at the center of the membrane phospholipid biosynthesis, and hence, PA levels are carefully controlled to maintain lipid homeostasis. PA is mostly generated by the phospholipase D (PLD)-catalyzed hydro- lysis of phosphatidylcholine. PLD1 and PLD2 catalyze the hydrolysis of phosphatidylcholine to PA and choline [10,11]. PA is also gener- ated by diacylglycerol kinases and lysophosphatidic acid acyl- transferases, which are central to phospholipid biosynthesis [12]. Studies have demonstrated the importance of PA in the stability and activity of mTOR complexes [12e14]. The responsiveness of mTOR to PA was shown to evolve as a means for sensing lipid precursors for membrane biosynthesis prior to cell growth and division [12]. As PA is involved in the stability and activity of mTOR complexes, we investigated whether PA mediates 1,25(OH)2D3- induced human myeloid cell differentiation.
C/EBPb, a member of the CCAAT/enhancer-binding protein
family, is a key transcription factor regulating human myeloid cells [15,16]. The treatment of human myeloid HL-60 cells with 12-O tetradecanoylphorbol-13-acetate (TPA; also known as phorbol 12- myristate 13-acetate, PMA) was shown to strongly induce the expression of C/EBPb mRNA [17]. In addition, the combination of PMA and lipopolysaccharide (LPS) was found to enhance C/EBPb promoter activity, resulting in an increase in the expression levels of C/EBPb mRNA and protein in human myeloid U937 cells [15]. Our previous study also showed that the expression of C/EBPb was increased in response to 1,25(OH)2D3 treatment of PMA- differentiated U937 cells [18]. Here, we demonstrate that 1,25(OH)2D3 induces the differentiation of human myeloid cells through the mTOR signaling pathway.

2. Materials and methods

2.1. Cell lines and culture conditions

Human myeloid cell lines, U937 and THP-1, were received from the Hospital for Rheumatic Diseases lab (Hanyang University, Seoul, Korea) and maintained in Roswell Park Memorial Institute (RPMI)- 1640 or Dulbecco’s modiﬁed Eagle’s minimum essential medium (Life Technologies, Carlsbad, CA) supplemented with 10% fetal bovine serum, 100 U/mL penicillin G sodium, 100 mg/mL strepto- mycin sulfate, and 0.25 mg/ml amphotericin B. For differentiation into macrophage-like cells, U937 and THP-1 cells were treated with 20 and 2 nM PMA, respectively, and allowed to adhere for 24 h. The differentiated cells were washed with phosphate-buffered saline
(PBS).

2.2. Reagents

The compounds 1,25(OH)2D3 and PMA were purchased from Sigma-Aldrich (St. Louis, MO). A stock solution of 1,25- dihydroxyvitamin D3 was prepared in ethanol. PP242 was pur- chased from Calbiochem (Philadelphia, PA, USA) and its stock so- lution was prepared in dimethyl sulfoxide (DMSO). We obtained 5-

ﬂuoro-2-indolyl des-chlorohalopemide (FIPI) from Sigma-Aldrich (St Louis, MO, USA) and Torin1, from Selleckchem (Houston, TX, USA). Antibodies against anti-phospho-S6K (9234), anti-S6K (2708), anti-phospho-AKT (4060), anti-AKT (4685), anti-Raptor (2280), and anti-Rictor (2114) were supplied by Cell Signaling (Danvers, MA, USA). Anti-C/EBPb (sc-150) was procured from Santa Cruz (Santa Cruz, CA, USA).

2.3. Small-interfering RNA (siRNA) transfection

PMA-differentiated THP-1 cells were transfected with Raptor, Rictor, or scrambled (negative) control siRNA using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer’s instruction. Protein expression of Raptor or Rictor was assessed with immuno- blotting after 24 h from transfection. The coding strand sequences of siRNAs are as follows: Raptor #1, 50-CCUCACUUUAUUUCCA-30; Raptor #2, 50-CGAGAUUGGACGACCAAAU-30; Rictor #1, 50-CUCUU- GAUAUCCUCGAUGA-30; and Rictor #2, 50-CACCUUCUUAAU- CAUCCAA-30.

2.4. Immunoblotting

Cell lysates were obtained using a lysis buffer (50 mM Tris, pH 7.5, 150 mM sodium chloride [NaCl], 1% NP-40, 0.5% Na- deoxycholate, protease inhibitor cocktail) and centrifuged at 13,000 rpm for 10 min at 4 ◦C. The protein concentration in extracts was quantiﬁed using the Bradford assay (Bio-Rad, Hercules, CA). Cell lysate was fractionated on 10% polyacrylamide gels with so- dium dodecyl sulfate polyacrylamide gel electrophoresis (SDS- PAGE), and the separated protein bands were transferred onto polyvinylidene ﬂuoride membranes (Millipore, Billerica, MA). The membranes were incubated with speciﬁc antibodies. Enhanced chemiluminescence (ECL) western blotting detection reagents (Amersham Biosciences) were used to detect the protein bands. For quantitative analysis of protein expression, the optical densities of the bands were measured using ImageJ software (Rasband, W.S., Image J, U. S. National Institutes of Health, Bethesda, Maryland, USA).

2.5. Flow cytometry

Cells were incubated with phycoerythrin (PE)-labeled CD14 antibody for 20 min in a cold room. After washing, the cells were suspended in PBS, and the percentage of CD14þ cells was assessed with FACSCalibur™ (Becton-Dickinson, San Jose, CA, USA). For the measurement of CD14 expression in PMA-differentiated cells, 10,000 U937 and THP-1 cells were analyzed for the forward scatter as the measure of cell size and FL-2/PE as the measure of the CD14þ population.

2.6. Morphological change

The PMA-differentiated U937 and THP-1 cells were treated with 1,25(OH)2D3 and observed under an inverted microscope (Eclipse 80i, Nikon, Tokyo, Japan).

2.7. Statistical analysis

The data are expressed as the mean ± standard deviation (SD) of three or more independent experiments. Statistical analysis was performed using the two-tailed Student’s t-test for paired data. P < 0.05 was considered statistically signiﬁcant.

3. Results

3.1. 1,25(OH)2D3 induces differentiation of human myeloid cells through the mTOR signaling pathway

As mTOR is involved in myeloid cell differentiation [4,5], we investigated whether the mTOR signaling pathway mediates the 1,25(OH)2D3-induced differentiation of human myeloid cells. First, the 1,25(OH)2D3-induced differentiation was assessed in PMA- stimulated U937 and THP-1 cells. As shown in Fig. 1A, the treat- ment with 1,25(OH)2D3 increased the expression of CD14, a marker of myeloid cell differentiation, in PMA-differentiated U937 and THP-1 cells. Furthermore, 1,25(OH)2D3 treatment also increased the phosphorylation of S6K (at Thr389) and AKT (at Ser473), indicative of the activation of both mTORC1 and mTORC2 (Fig. 1B).
We examined whether the 1,25(OH)2D3-induced differentiation was regulated by the mTOR signaling pathway. The increase in CD14 expression by 1,25(OH)2D3 was suppressed by dual mTOR inhibitors, PP242 and Torin1 (Fig. 1C), suggesting that the mTOR signaling pathway was a critical mediator of differentiation induced by 1,25(OH)2D3 in PMA-treated U937 and THP-1 cells. The activa- tion of both mTORC1 and mTORC2 was suppressed by PP242 and

Torin1, as evident from the phosphorylation of S6K at Thr 389 and AKT at Ser 473, respectively (Fig. 1D).
The changes in cellular morphology can be monitored during the differentiation of human myeloid cells such as HL-60 and HL-
525 [19]. Upon differentiation, the cells strongly adhere to the surfaces and spread. As shown in Fig. 1E, the morphological ex- amination of PMA-differentiated U937 and THP-1 cells showed that the cells exhibited increased adherence to the dishes and could spread in the presence of 1,25(OH)2D3. Morphological changes induced by 1,25(OH)2D3 could be reversed by mTOR inhibitors, PP242 and Torin1. Altogether, these results suggest that the mTOR signaling pathway is activated by 1,25(OH)2D3 and is essential for the 1,25(OH)2D3-mediated differentiation of human myeloid cells.

3.2. Both mTORC1 and mTORC2 are important for 1,25(OH)2D3einduced differentiation

We investigated whether Raptor and Rictor, the major compo- nents of mTORC1 and mTORC2, respectively, regulate human myeloid cell differentiation induced by 1,25(OH)2D3. The expres- sion of Raptor and Rictor was shown to increase during the dif- ferentiation of HL60 cells [4]. As shown in Fig. 2A, the 1,25(OH)2D3-

Fig. 1. Inhibition of mTOR signaling pathway suppresses the differentiation of human myeloid cells. U937 and THP-1 cells were treated with 20 and 2 nM PMA, respectively, to induce their differentiation into macrophages. Differentiated cells were washed with PBS. (A) 1,25(OH)2D3 was used at 50 nM concentration for 24 h. Cells were stained with PE- CD14, and CD14 expression was analyzed with ﬂow cytometry. The representative results of three separate experiments are shown. Data represent the mean ± SD of triplicate measurements, representative of three independent experiments. (B) U937 and THP-1 cells were treated with 20 and 30 nM of 1,25(OH)2D3, respectively. Phosphorylation of S6K and AKT was analyzed with immunoblotting. b-Actin was included as a loading control. (C) Cells were pretreated with PP242 (200 nM) or Torin1 (200 nM) for 1 h before treatment with 1,25(OH)2D3. Cells were stained with PE-CD14, and CD14 expression was analyzed with ﬂow cytometry. (D) Changes in cellular morphology were observed under a light microscope. (E) U937 cells were pretreated with PP242 (200 nM) or Torin1 (200 nM) for 1 h before treatment with 1,25(OH)2D3. Cell lysates were analyzed for S6K and AKT phosphorylation with immunoblotting.

Fig. 2. Silencing of Raptor or Rictor inhibited the differentiation induced by 1,25(OH)2D3 in PMA-differentiated THP-1 cells. THP-1 cells were treated with 2 nM PMA for their differentiation into macrophages. PMA-differentiated cells were washed with PBS. (A) Cells were transfected with 100 pmol of control, Raptor, or Rictor siRNA and treated with 50 nM of 1,25(OH)2D3 for 24 h. Cells were stained with PE-CD14, and CD14 expression was analyzed with ﬂow cytometry. (B) Cellular morphological changes were detected using a microscope. (C) Cells were transfected with 100 pmol of control, Raptor, or Rictor siRNA. The expression of Raptor and Rictor protein was analyzed with immunoblotting. b-Actin was used as a loading control.

mediated increase in CD14 expression was suppressed after the silencing of either Raptor or Rictor in PMA-differentiated THP- 1 cells, indicative of the involvement of both mTORC1 and mTORC2. We also observed that the 1,25(OH)2D3einduced morphological changes were reversed after the silencing of Raptor or Rictor expression (Fig. 2B). Thus, 1,25(OH)2D3 induces human myeloid cell differentiation via the mTOR signaling pathway in the presence of both mTORC1 and mTORC2.

3.3. PA is required for myeloid cell differentiation induced by 1,25(OH)2D3

Recent studies have revealed the important role of PA in the stability and, hence, the activity of both mTORC1 and mTORC2 complexes [10e12]. PA production can be suppressed with 1- butanol or the PLD inhibitor, FIPI. To investigate whether PA plays a role in CD14 upregulation by 1,25(OH)2D3, we depleted the expression of PA. In PMA-differentiated U937 and THP-1 cells, the upregulation of CD14 by 1,25(OH)2D3 was attenuated by 1-butanol or FIPI (Fig. 3A), indicative of the PA-mediated regulation of CD14 expression. In addition, the 1,25(OH)2D3einduced morphological changes were reversed by 1-butanol or FIPI (Fig. 3B). AKT phos- phorylation at Ser 473 was also suppressed after 1-butanol or FIPI treatment (Fig. 3C). In conclusion, 1,25(OH)2D3einduced differen- tiation is regulated by PA in human myeloid cells.

3.4. The mTOR signaling pathway triggered by 1,25(OH)2D3 upregulates C/EBPb expression

C/EBPb is a critical regulator of human myeloid cells [15] and 1,25(OH)2D3 induces CD14 upregulation through the transcrip- tional activation of C/EBPb in HL-60, U937, and THP-1 cells [20]. Therefore, we hypothesized that C/EBPb was activated through the mTOR signaling pathway to drive human myeloid cell differentia- tion in response to 1,25(OH)2D3 treatment. The protein level of C/ EBPb was assessed in PMA-differentiated U937 cells in response to 1,25(OH)2D3 treatment. As shown in Fig. 4A, the treatment with 1,25(OH)2D3 resulted in the upregulation of the protein expression of C/EBPb. We also examined whether C/EBPb upregulation by 1,25(OH)2D3 was mediated through the mTOR signaling pathway. C/EBPb upregulation by 1,25(OH)2D3 was inhibited by PP242 or Torin1 (Fig. 4B), suggestive of the critical role of the mTOR signaling pathway in C/EBPb upregulation mediated by 1,25(OH)2D3 in PMA- differentiated U937 cells. PA is necessary for C/EBPb expression upregulation, as 1-butanol or FIPI reduced the induction of C/EBPb protein expression (Fig. 4C). To further conﬁrm the role of mTOR signaling pathway in 1,25(OH)2D3-induced C/EBPb upregulation,

the expression of Raptor or Rictor was knocked down with RNA interference. While C/EBPb expression was upregulated by 1,25(OH)2D3, the knockdown of Raptor or Rictor expression reversed the 1,25(OH)2D3-induced upregulation of C/EBPb expres- sion (Fig. 4D). Altogether, these results indicate that 1,25(OH)2D3 induces C/EBPb upregulation via the mTOR signaling pathway in the presence of both mTORC1 and mTORC2.

4. Discussion

Human myeloid cells may differentiate into monocyte-like cells after treatment with 1,25(OH)2D3, indicating that 1,25(OH)2D3 may be useful in differentiation therapy. However, the intracellular signaling mechanism underlying 1,25(OH)2D3-induced differenti- ation of human myeloid cells remains elusive. In this report, we investigated the involvement of the mTOR signaling pathway in the differentiation of human myeloid cells following 1,25(OH)2D3 exposure. We found that 1,25(OH)2D3 activated the mTOR signaling pathway in human myeloid cells, as evident from the increase in the phosphorylation of S6K (at Thr 389) and AKT (at Ser 473), the key mediators of mTORC1 and mTORC2, respectively. The treat- ment of PMA-induced differentiated U937 and THP-1 cells with 1,25(OH)2D3 also resulted in an increase in the expression of CD14 and induced morphological changes typical of differentiated myeloid cells. These effects were suppressed by the dual mTOR inhibitors, PP242 and Torin1. The upregulation of CD14 expression by 1,25(OH)2D3 was prevented by the silencing of Raptor or Rictor expression in PMA-differentiated THP-1 cells. Incubation of cells with 1-butanol or the PLD inhibitor FIPI resulted in the inhibition of PA production and reversion of CD14 upregulation and

Fig. 3. Involvement of PA in 1,25(OH)2D3-induced human myeloid cell differentiation. (A) U937 and THP-1 cells were treated with 50 nM 1,25(OH)2D3 for 24 h. Cells were pre- treated with 0.5% of 1-butanol or FIPI and stained with PE-CD14. CD14 expression was analyzed with ﬂow cytometry. (B) U937 and THP-1 cells were treated with 20 and 30 nM of 1,25(OH)2D3, respectively. Cells were pre-treated with 0.5% of 1-butanol or FIPI for 1 h before treatment with 1,25(OH)2D3. Morphological changes were detected with microscopy. (C). In U937 cells, lysates were analyzed for the expression of S6K and AKT with immunoblotting. b-Actin was used as a loading control.

Fig. 4. Upregulation of C/EBPb expression by 1,25(OH)2D3-induced mTOR signaling pathway. U937 and THP-1 cells were treated with 20 and 2 nM PMA, respectively, for their differentiation into macrophages. Differentiated cells were washed with PBS. (AeC) The PMA-differentiated U937 cells were treated with 1,25(OH)2D3 at 5e20 nM concentrations for 2e24 h. Cell lysates were analyzed for C/EBPb expression with immunoblotting. b-Actin was used as a loading control (A). Cells were pretreated with PP242 (200 nM), Torin1 (200 nM), 0.5% of 1-butanol, or FIPI for 1 h before treatment with 1,25(OH)2D3. Cell lysates were analyzed for C/EBPb expression with immunoblotting (BeC). (D) Cells were treated with 30 nM of 1,25(OH)2D3 for 24 h and transfected with control, Raptor, or Rictor siRNA (100 pmol). The protein expression of C/EBPb, Raptor, and Rictor was analyzed with immunoblotting. b-Actin was used as a loading control (D).

morphological changes induced by 1,25(OH)2D3. Finally, the 1,25(OH)2D3-induced upregulation of C/EBPb, a key transcription factor involved in myeloid differentiation, was prevented through the inhibition of mTOR or PA production. Based on these evidences, we propose that 1,25(OH)2D3 may induce the differentiation of human myeloid cells via the mTOR signaling pathway.
mTOR exists as two distinct evolutionarily conserved com- plexes, mTORC1 and mTORC2 [19]. The silencing of Rictor expres- sion by siRNA was found to inhibit the reducing ability of nitro blue- tetrazolium (NBT) following 1,25(OH)2D3-induced cell differentia- tion [4]. mTORC2 regulated the activity of both AKT and PKC which are involved in cellular functions such as NBT reduction as well as the esterase activity associated with myeloid cell differentiation [4]. In addition, Jacinto et al. [21] reported that the activation of mTOR signaling pathway may lead to actin polymerization and spreading in NIH3T3 ﬁbroblast cells. Rapamycin insensitivity observed by these authors suggests the involvement of mTORC2, but not mTORC1, in the organization of actin cytoskeleton. In U937 cells, the inhibition of mTORC1 by rapamycin had no preventive effect on the upregulation of CD14 expression induced by 1,25(OH)2D3 (Fig. 1C). However, the inhibition of both mTORC1 and mTORC2 by the dual mTOR inhibitors PP242 and Torin1 prevented the upre- gulation of CD14 expression. In our study, both mTORC1 and mTORC2 appeared to be required for the differentiation of myeloid THP-1 cells, as the silencing of either Raptor or Rictor resulted in the inhibition of the upregulation of CD14 expression and prevented the morphological changes associated with myeloid cell differen- tiation. These differences between U937 and THP-1 cells may be attributed to the different origins and maturation stages of these

cells. U937 cells are derived from tissues (histocytic lymphoma) and are at a mature stage, while THP1 cells are of blood leukemia origin and at a less mature stage [22e24]. Thus, mTORC2 plays a more important role than mTORC1 in 1,25(OH)2D3-induced differ- entiation of U937 cells.
In the present study, the inhibition of PA production resulted in the suppression of upregulated CD14 expression and morpholog- ical changes induced by 1,25(OH)2D3. The reduced production of PA by 1-butanol or FIPI treatment suppressed the phosphorylation of both S6K and AKT, indicative of the role of PA in the activation of both mTORC1 and mTORC2 [25,26]. However, PA inhibitors sup- pressed the phosphorylation of AKT (at Ser 473), but not S6K (at Thr 389), in U937 cells, suggesting that PA depletion mostly affected mTORC2 activation.
C/EBPb regulates the expression of a variety of differentiation-
associated genes, including CD14 [16,27]. In our results, the impairment of the mTOR signaling pathway by mTOR inhibitor or reduction in PA production prevented the increase in C/EBPb pro- tein expression, suggesting that mTOR induced C/EBPb expression during the differentiation of human myeloid cells. C/EBPb exists in three different isoforms, namely, LAP* (full-length protein), LAP (a 21 amino acid truncation at the N terminus), and LIP (a large N- terminal truncation) [28]. The three C/EBPb isoforms, LAP*, LAP, and LIP, are generated by alternative initiation of protein translation from a single transcript. Numerous experiments have shown that LAP* and LAP isoforms are activators of gene transcription. In contrast, the LIP form lacks the transactivation domain and func- tions as a dominant-negative form to suppress gene activation [28,29]. The functional differences between LAP* and LAP are incompletely understood. The differentiation of myeloid cells is characterized by a signiﬁcant upregulation in the expression of C/ EBPb, especially the LAP* and LAP forms [15]. The upregulation in the expression of both LAP* and LAP by 1,25(OH)2D3 was sup- pressed after the silencing of Rictor and, to some extent, Raptor.
In conclusion, our results showed that 1,25(OH)2D3 enhances the differentiation of human myeloid cells through the mTOR signaling pathway. A better understanding of the differentiation of human myeloid cells by the mTOR signaling pathway would facil- itate proper clinical applications of mTOR regulators such as 1,25(OH)2D3 for the treatment of myeloproliferative diseases, including leukemia.

Funding

This work was supported by Basic Science Research Pro-gram through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology (2017R 1D 1A 1B03028389 to Jong Dae Ji).
Transparency document
Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.09.100

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