OLFM4 was identified initially as a gene highly induced in myeloid stem cells by G-CSF treatment. A bioinformatics method using a global meta-analysis of microarray data predicted that OLFM4 would be associated with specific granules in human neutrophils. Subcellular fractionation of peripheral blood neutrophils demonstrated complete colocalization of OLFM4 with the specific granule protein NGAL, and stimulation of neutrophils with PMA resulted in corelease of NGAL and OLFM4, proving that OLFM4 is a genuine constituent of neutrophil-specific granules. In accordance with this, OLFM4 mRNA peaked at the MY/MM stage of maturation. OLFM4 was, however, present in only 20-25% of peripheral blood neutrophils, as determined by immunocytochemistry and flow cytometry, whereas mRNA for OLFM4 was present in all MY/MM, indicating post-transcriptional regulation as a basis for the heterogeneous expression of OLFM4 protein.
Whereas neutrophil elastase, cathepsin G, and proteinase 3 have been known as granule-associated serine proteases of neutrophils for decades, a fourth member, called neutrophil serine protease 4 (NSP4), was just recently described and provisionally characterized. In this study, we identified NSP4 as a novel azurophil granule protein of neutrophils by Western blot analyses of subcellular fractions as well as by RT-PCR analyses of neutrophil precursors from human bone marrow. The highest mRNA levels were observed in myeloblasts and promyelocytes, similar to myeloperoxidase, a marker of azurophil granules. To determine the extended sequence specificity of recombinant NSP4, we used an iterative fluorescence resonance energy transfer-based optimization strategy. In total, 142 different peptide substrates with arginine in P1 and variations at the P1', P2', P3, P4, and P2 positions were tested. This enabled us to construct an alpha(1)-proteinase inhibitor variant (Ile-Lys-Pro-Arg-/-Ser-Ile-Pro) with high specificity for NSP4. This tailor-made serpin was shown to form covalent complexes with all NSP4 of neutrophil lysates and supernatants of activated neutrophils, indicating that NSP4 is fully processed and stored as an already activated enzyme in azurophil granules. Moreover, cathepsin C was identified as the activator of NSP4 in vivo, as cathepsin C deficiency resulted in a complete absence of NSP4 in a Papillon-Lefevre patient. Our in-depth analysis of NSP4 establishes this arginine-specific protease as a genuine member of preactivated serine proteases stored in azurophil granules of human neutrophils
Smad4 is important in the TGF- pathway and required for transcriptional activation and inhibition of cell growth after TGF-1 stimulation. We demonstrate that miR-130a is differentially expressed during granulopoiesis and targets Smad4 mRNA. The transcript for Smad4 is present throughout neutrophil maturation, but Smad4 protein is undetectable in the most immature cells, where miR-130a is highly expressed. Two miR-130a binding sites were identified in the 3-untranslated region of the IntroductionMature neutrophils are generated in the bone marrow (BM) from myeloid precursor cells, the myeloblasts, which divide and mature along a tightly regulated path characterized by cessation of proliferation, nuclear condensation, and sequential acquisition of different types of granules, 1 collectively known as granulopoiesis. Granulopoiesis is controlled by intrinsic and extrinsic factors to ensure both a strict control of the stepwise maturation of the cells and to control the rate of production and release to meet the requirements for an adequate defense against microbial infections. 2 Under normal conditions, maturation of the neutrophil granulocyte precursors is accompanied by a progressive and differential expression of several transcription factors, 3 which regulate cell division 4,5 and formation of granules. 1 In case of an increased demand for neutrophils, the differentiation process can be adjusted by external stimuli such as G-CSF, 6 bacterial products, 7 or proinflammatory cytokines 8 that induce changes in the level and composition of the transcription factors that control cell proliferation and terminal differentiation.TGF- is a potent cytokine that affects many biologic functions such as proliferation, differentiation, and apoptosis, depending on the developmental state and type of cell. 9 It has been shown that TGF-1 inhibits proliferation in quiescent HSCs. 10 Members of the TGF- superfamily of growth factors, including TGF-s, bind to TGF- receptor I (TGFRI) and TGFRII expressed on the cell surface. Binding of ligands to TGFRII induces formation of receptor complexes which phosphorylate and activate TGFRI. The active form of TGFRI then recruits and phosphorylates the receptor-activated Smads (R-Smad). Phosphorylated R-Smads assemble and form a hetero-oligomeric complex with a commonSmad (Co-Smad). This complex accumulates in the nucleus and binds to specific Smad-binding elements in the promoters of TGF--responsive genes where it recruits coactivators and corepressors and thus exerts both a positive and a negative regulation of target gene expression. 11,12 Smad2 and Smad3 are R-Smads that transmit TGF-1/activin-induced signals, whereas Smad1, Smad5, and Smad8 are R-Smads that transmit bone morphogenetic proteininduced signals. Smad6 and Smad7 are inhibitory Smads that regulate TGF-1 signaling negatively by competitive binding to the TGF- receptors or to the Smad4 is the only known Co-Smad in mammals and thus plays a central role in the TGF--signaling pathway because all RSmads must form a...
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