Pathways of retinoid synthesis in mouse macrophages and bone marrow cells

Abstract

In vivo pathways of natural retinoid metabolism and elimination have not been well characterized in primary myeloid cells, even though retinoids and retinoid receptors have been strongly implicated in regulating myeloid maturation. With the use of a upstream activation sequence-GFP reporter transgene and retrovirally expressed Gal4-retinoic acid receptor α in primary mouse bone marrow cells, we identified 2 distinct enzymatic pathways used by mouse myeloid cells ex vivo to synthesize retinoic acid receptor α ligands from free vitamin A metabolites (retinyl acetate, retinol, and retinal). Bulk Kit(+) bone marrow progenitor cells use diethylaminobenzaldehyde-sensitive enzymes, whereas bone marrow-derived macrophages use diethylaminobenzaldehyde-insensitive enzymes to synthesize natural retinoic acid receptor α-activating retinoids (all-trans retinoic acid). Bone marrow-derived macrophages do not express the diethylaminobenzaldehyde-sensitive enzymes Aldh1a1, Aldh1a2, or Aldh1a3 but instead, express Aldh3b1, which we found is capable of diethylaminobenzaldehyde-insensitive synthesis of all trans-retinoic acid. However, under steady-state and stimulated conditions in vivo, diverse bone marrow cells and peritoneal macrophages showed no evidence of intracellular retinoic acid receptor α-activating retinoids, despite expression of these enzymes and a vitamin A-sufficient diet, suggesting that the enzymatic conversion of retinal is not the rate-limiting step in the synthesis of intracellular retinoic acid receptor α-activating retinoids in myeloid bone marrow cells and that retinoic acid receptor α remains in an unliganded configuration during adult hematopoiesis.

Keywords: ATRA; Aldh3b1; aldehyde dehydrogenase; retinoid receptor.

Figure 1.. Activation of Gal4-RARA by natural retinoids.

(A) 293T cells were transfected, as indicated; treated with liarozole, DEAB, or ATRA; and analyzed for GFP expression after 72 h. (B) Model of stepwise ATRA synthesis and effect of inhibitors. (C and D) Activation of UAS-GFP reporter by MSCV-Gal4-RARA-IC in Kit⁺ progenitor cells. (E and F) Activation of UAS-GFP reporter by MSCV-Gal4-RARA-IC in BMMφ. In each case, cells were prepared, transduced with MSCV-Gal4-RARA-IC, and then treated as indicated for 72 h before analysis of GFP. Error bars represent sd of triplicate experiments.

Figure 2.. Differential sensitivity of pathways leading to natural Gal4-RARA-activating ligands in Kit⁺ progenitor cells and in BMMφ.

(A and B) Kit⁺ progenitor cells and BMMφ were transduced with MSCV-Gal4-RARA-IC retrovirus and treated as indicated for 72 h before analysis of GFP within cells expressing mCherry. (C) BMMφ were transduced with MSCV-Gal4-RARA-IC and treated with WIN 18466, as indicated for 72 h before analysis of GFP within cells expressing mCherry. (D and E) BMMφ were transduced with MSCV-Gal4-RARA-IC and treated with 300 nM of indicated retinoids for 72 h before analysis of GFP within cells expressing mCherry. Error bars represent sd of triplicate experiments.

Figure 3.. Differentially expressed enzyme transcripts in Kit⁺ progenitors and BMMφ.

(A) Semiquantitative RT-PCR analysis of Aldh1a1, Aldh1a2, and Aldh1a3 and 2 control transcripts, ornithine decarboxylase antizyme 1 (Oaz1) and Gapdh, in Kit⁺ progenitor cells and BMMφ. (B) Affymetrix expression array of Kit⁺ progenitor cells vs. BMMφ. (C) Affymetrix expression results for 5 transcripts with differential expression between Kit⁺ progenitor cells and BMMφ. Data normalized to chip mean of 1500. (D) Quantitative RT-PCR analysis of Aldh genes in Kit⁺ progenitor cells vs. BMMφ. In all studies, error bars represent sd of triplicate experiments.

Figure 4.. Aldh3b1 expression associated with Gal4-RARA activation and ATRA synthesis.

(A) Stable 293T UGN/FAP cells were transiently transfected with indicated expression vectors, treated as indicated, and analyzed, 72 h later. (B) 293T cells were transfected with UAS-GFP reporter, Gal4-RARA expression plasmid, and indicated Aldh expression plasmids and treated as indicated. pBS, pBluescript. (C) Stable 293T UGN/FAP cells were transiently transfected with Aldh3b1 expression plasmid and treated with liarozole or with RARA antagonists, as indicated. (D) 293T cells were transiently transfected with the UAS-GFP reporter and expression plasmids for Aldh3b1, Gal4-RARA, or Gal4-RXRA, as indicated. (E) 293T cells were transfected with UAS-GFP reporter plasmids, Gal4-RARA expression plasmids, and indicated Aldh3b1 plasmids. Mu, Murine; Hu, human. Cells were treated with or without liarozole for 72 h and assessed by flow cytometry. Error bars indicate sd of triplicate experiments.

Figure 5.. Aldh3b1 activity.

(A) Mouse (m)Aldh3b1 and human (h)ALDH3B1 were transfected into 293T cells and ALDH activity assessed in total protein lysates using octanal as a substrate. (B) Endogenous mouse Aldh3b1 activity was assessed in total protein lysates from BMMφ and Kit⁺ progenitor cells using octanal as substrate (Lineweaver-Burk plot is inset). Error bars indicate sd of duplicates. (C) MS determination of ATRA concentrations in cell-culture media from 293T stable UGN/FAP cells transfected with Aldh3b1 and treated with DEAB or ATRA. (D) MS of ATRA concentrations in total cells transfected in C. (E) Schema of knockdown Aldh3b1 in BMMφ. (F) Western blot of BMMφ transfected with siRNA targeting Aldh3b1. HPRT, Hypoxanthine phosphoribosyltransferase. (G) Percentage of GFP⁺ BMMφ after knockdown Aldh3b1. (H) GFP MFI of BMMφ after knockdown Aldh3b1. Error bars indicate sd of triplicate measurements.

Figure 6.. In vivo analysis of natural retinoids in myeloid cells.

UAS-GFP bone marrow Kit⁺ cells were transduced with MSCV-Gal4-RARA-IC and transplanted into lethally irradiated recipient mice. After 6 wk engraftment, mice were untreated or treated with ATRA by daily gavage for 3 d. Peritoneal macrophages and bone marrow monocytes (Gr1+CD11b⁺) were then analyzed for GFP expression within cells that express mCherry. Two representative mice are shown, from a total of 5 mice analyzed with each treatment. FL1/3, Fluorescence 1/3. INT LOG, Integrated log scale.

Figure 7.. Stra6 in BMMφ.

(A) Semiquantitative RT-PCR of Stra6 and Oaz1 in RNA extracted from spleen, liver, kidney, total bone marrow (BM) cells, and Kit⁺ progenitor cells and in BMMφ. PCR was performed with 50 ng total RNA starting material/well and 34 cycles. Expected fragment size Stra6, 379 bp; Oaz1, 332 bp. (B) Schema of BMMφ transduction and evaluation. (C and D) After transduction with MSCV-STRA6-IRES-Cerulean, Cerulean-negative BMMφ were transduced with Gal4-RARA-IC and treated with or without mouse serum for 48 h before evaluation. (E and F) Cerulean-positive BMMφ were transduced with MSCV-Gal4-RARA-IC and treated with or without mouse serum for 48 h before evaluation. Representative data of 2 independent experiments.

Figure 8.. Hematopoietic stress does not induce natural RARA ligand synthesis in hematopoietic cells.

UAS-GFP bone marrow cells were transduced with MSCV-Gal4-RARA-IC and transplanted into recipient mice. After engraftment, mice were treated with 5FU, phenylhydrazine, G-CSF, Ova-alum, or neuraminidase. Bone marrow cells and peritoneal macrophages collected after 2 d (neuraminidase), 3 d (phenylhydrazine, G-CSF, and Ova-alum), or 9 d (5FU). Similar results were observed on d 3, 6, and 12 after 5FU. Representative data of at least 3 separately treated recipient mice.