Many microRNAs (miRNAs) are evolutionarily conserved and have intriguing expression patterns. Tissue and/or time-specific expressions of some miRNAs are presumably controlled by unique cis-acting regulatory elements that coevolved with the miRNA sequences. Exploiting bioinformatics, we identified several miRNAs whose primary transcripts could be regulated by conserved genomic elements proximal to their transcription start sites. Such miRNAs include microRNA-223 (miR-223), which is reportedly controlled by a unique regulatory mechanism during granulopoiesis. Here, we define a mechanism distinct from that previously proposed to regulate miR-223 expression. We find that the mir-223 gene resembles a "myeloid gene" and might be driven by the myeloid transcription factors, PU.1 and C/EBPs. This mechanism is specified by the conserved proximal cis-regulatory element and might be common among different species. Hence, it needs to be considered that two distinct mechanisms that would play critical roles in myeloid functions and differentiation are actually concerned with the regulation of miR-223.

Recurrent mutations in the gene encoding additional sex combs-like 1 (ASXL1) are found in various hematologic malignancies and associated with poor prognosis. In particular, ASXL1 mutations are common in patients with hematologic malignancies associated with myelodysplasia, including myelodysplastic syndromes (MDSs), and chronic myelomonocytic leukemia. Although loss-of-function ASXL1 mutations promote myeloid transformation, a large subset of ASXL1 mutations is thought to result in stable truncation of ASXL1. Here we demonstrate that C-terminal-truncating Asxl1 mutations (ASXL1-MTs) inhibited myeloid differentiation and induced MDS-like disease in mice. ASXL1-MT mice displayed features of human-associated MDS, including multilineage myelodysplasia, pancytopenia, and occasional progression to overt leukemia. ASXL1-MT resulted in derepression of homeobox A9 (Hoxa9) and microRNA-125a (miR-125a) expression through inhibition of polycomb repressive complex 2-mediated (PRC2-mediated) methylation of histone H3K27. miR-125a reduced expression of C-type lectin domain family 5, member a (Clec5a), which is involved in myeloid differentiation. In addition, HOXA9 expression was high in MDS patients with ASXL1-MT, while CLEC5A expression was generally low. Thus, ASXL1-MT-induced MDS-like disease in mice is associated with derepression of Hoxa9 and miR-125a and with Clec5a dysregulation. Our data provide evidence for an axis of MDS pathogenesis that implicates both ASXL1 mutations and miR-125a as therapeutic targets in MDS.

Leukocyte mono-immunoglobulin (Ig)–like receptor 5 (LMIR5)/CD300b is a DAP12-coupled activating receptor predominantly expressed in myeloid cells. The ligands for LMIR have not been reported. We have identified T cell Ig mucin 1 (TIM1) as a possible ligand for LMIR5 by retrovirus-mediated expression cloning. TIM1 interacted only with LMIR5 among the LMIR family, whereas LMIR5 interacted with TIM4 as well as TIM1. The Ig-like domain of LMIR5 bound to TIM1 in the vicinity of the phosphatidylserine (PS)-binding site within the Ig-like domain of TIM1. Unlike its binding to TIM1 or TIM4, LMIR5 failed to bind to PS. LMIR5 binding did not affect TIM1- or TIM4-mediated phagocytosis of apoptotic cells, and stimulation with TIM1 or TIM4 induced LMIR5-mediated activation of mast cells. Notably, LMIR5 deficiency suppressed TIM1-Fc–induced recruitment of neutrophils in the dorsal air pouch, and LMIR5 deficiency attenuated neutrophil accumulation in a model of ischemia/reperfusion injury in the kidneys in which TIM1 expression is up-regulated. In that model, LMIR5 deficiency resulted in ameliorated tubular necrosis and cast formation in the acute phase. Collectively, our results indicate that TIM1 is an endogenous ligand for LMIR5 and that the TIM1–LMIR5 interaction plays a physiological role in immune regulation by myeloid cells.

Leukocyte mono-Ig-like receptor 3 (LMIR3) is an inhibitory receptor mainly expressed in myeloid cells. Coengagement of FcεRI and LMIR3 impaired cytokine production in bone marrow-derived mast cells (BMMCs) induced by FcεRI crosslinking alone. Mouse LMIR3 possesses five cytoplasmic tyrosine residues (Y241, Y276, Y289, Y303, Y325), among which Y241 and Y289 (Y241/289) or Y325 fit the consensus sequence of ITIM or immunotyrosine-based switch motif (ITSM), respectively. The inhibitory effect was abolished by the replacement of Y325 in addition to Y241/289 with phenylalanine (Y241/189/325/F) in accordance with the potential of Y241/289/325 to cooperatively recruit Src homology region 2 domain-containing phosphatase 1 (SHP)-1 or SHP-2. Intriguingly, LMIR3 crosslinking alone induced cytokine production in BMMCs expressing LMIR3 (Y241/276/289/303/325F) mutant as well as LMIR3 (Y241/289/325F). Moreover, coimmunoprecipitation experiments revealed that LMIR3 associated with ITAM-containing FcRγ. Analysis of FcRγ-deficient BMMCs demonstrated that both Y276/303 and FcRγ played a critical role in the activating function of this inhibitory receptor. Importantly, LMIR3 crosslinking enhanced cytokine production of BMMCs stimulated by LPS, while suppressing production stimulated by other TLR agonists or stem cell factor. Thus, an inhibitory receptor LMIR3 has a unique property to associate with FcRγ and thereby functions as an activating receptor in concert with TLR4 stimulation.

Thiazolidinediones (TZDs) improve insulin resistance by activating a nuclear hormone receptor, peroxisome proliferator-activated receptor γ (PPARγ). However, the use of TZDs is associated with plasma volume expansion through a mechanism that remains to be clarified. Here we showed that TZDs rapidly stimulate sodium-coupled bicarbonate absorption from the renal proximal tubule in vitro and in vivo. TZD-induced transport stimulation is dependent on PPARγ-Src-EGFR-ERK and observed in rat, rabbit and human, but not in mouse proximal tubules where Src-EGFR is constitutively activated. The existence of PPARγ-Src-dependent nongenomic signaling, which requires the ligand-binding ability, but not the transcriptional activity of PPARγ, is confirmed in mouse embryonic fibroblast cells. The enhancement of the association between PPARγ and Src by TZDs supports an indispensable role of Src in this signaling. These results suggest that the PPARγ-dependent nongenomic stimulation of renal proximal transport is also involved in TZD-induced volume expansion.