A gene that encodes mouse mast cell protease (mMCP) 7 (also known as mouse mast cell tryptase 2) was isolated by genomic cloning with a cDNA that encodes mMCP-6, a tryptase in serosal mast cells. cDNAs encoding mMCP-7 were isolated from a bone-marrow-derived mast cell cDNA library. The mMCP-7 gene spans 2.3 kilos and contains five exons rather than six, as found in the mMCP-6 and human mast cell yptase I genes. Comparison ofthe 5' end of the transcript with the genomic sequence ted that the region corresponding to the first intron in the mMCP-6 and human tryptase I genes Is not spliced during trnscription of mMCP-7 mRNA because of a point mutation at the intron 1 acceptor splice site; this results in a 5' untransated region of 195 nucleotides, which is longer than that of any other known mast cell-specific transcpt. mMCP-7 is 71-76% homologous with mMCP-6 and with dog and human mast cell tryptases, and it is the most acidic mast cell protease, with an overall net charge of -10. RNA blot analyses revealed that the mMCP-7 gene is transcribed in bone-marrow-derived mast cells but is not transcribed in mature serosal mast cells or in mucosal mast cell-enriched intestinal tissue of Trihinefla spinl-infected mice. Transcription of the mMCP-7 gene by differentiating bone-marrow-derived mast cells occurred within 1 week of bone-marrow culture but decreased dramatically after 3 weeks. Thus, the mMCP-7 gene displays a number of unusual structurl characteristics and is distinctive in its transient and selective expression in Immature mast cells maintained in interleukin 3-enriched medium.The secretory granules of mouse mast cells contain a family of serine proteases termed mouse mast cell protease (mMCP) 1 through mMCP-6 (1). mMCP-1 (2-4), mMCP-2 (5), mMCP4 (6), and mMCP-5 (7) have been designated as chymases because of the predicted chymotryptic-like substrate specificities of their translated gene products. mMCP-6 (8) has been designated a tryptase because of its predicted trypticlike substrate specificity. The mouse mast cell chymases have overall net charges at pH 7 from +2 to + 15, whereas mMCP-6 has an overall net charge of -5. Serosal mast cells selectively express mMCP-4, mMCP-5, and mMCP-6, whereas mucosal mast cells that proliferate in the intestines of helminth-infected mice selectively express mMCP-1 and mMCP-2. The mMCP-5 and mMCP-6 genes are transcribed in bone-marrow-derived mast cells (BMMCs) obtained by culturing bone-marrow cells in medium containing interleukin (IL) 3. Because the BMMCs reconstitute all populations of mast cells in vivo when injected into mast cell-deficient
As assessed by RNA blot analyses with genespecific probes, we report that the perivascular connective tissue mast ceOls (CTMCs) in the ear and skin of BALB/cJ mice contain abundant levels of the mouse mast cell protease (mMCP) 7 transcript, in addition to those protease transcripts present in their serosal mast cells (SMCs). High levels of the mMCP-7 transcript also were detected in the ears of WBB6F1/J-+/+, WCB6F1/J-+/+, WB/ReJ-+/+, and WC/ReJ-+/+ mice. However, the ears of these four strains and the SMCs from the WCB6F1/J-+/+ strain but not the BALB/cJ strain also contained high steady-state levels of the mMCP-2 transcript. The mMCP-2, mMCP-4, mMCP-5, mMCP-6, and mMCP-7 transcripts were not detected in the ears of mast-cell-deficient WBB6F1/J-W/Wv and WCB6F1/JSli/SId mice, indicating that mast cells were the source of these protease transcripts in the +/+ animals. When immunohistochemic*l analyses of serial sections of ear and skin from WBB6F1/J-+/+ mice were performed with anti-mMCP-2IgG and anti-mMCP-5 IgG, the perivascular CTMCs in these tissues were found to express both mMCP-2 and mMCP-5 in their granules. The prominent expression of mMCP-7 in constitutive perivascular CTMCs indicates that this mast cell has an extended protease phenotype relative to the SMCs of the same strains. Further, the perivascular CTMCs and SMCs of the +/+ strains differ from those in BALB/cJ mice in their prominent expression of mMCP-2.As assessed by protein sequencing and/or by cDNA cloning, mouse mast cells differentiated in vivo and in vitro express varied combinations of carboxypeptidase A (mMC-CPA) (1) and at least seven serine proteases [designated mouse mast cell protease (mMCP) 1 to mMCP-7] in their granules (2-11). The safranin+ staining serosal mast cells (SMCs) of the BALB/cJ mouse contain high steady-state levels of the transcripts that encode mMCP-4, mMCP-5, and mMCP-6 but not mMCP-1, mMCP-2, or mMCP-7. In contrast, the safranin-mucosal mast cells that proliferate in the intestine of helminth-infected BALB/cJ mice contain high steady-state levels of the mMCP-1 and mMCP-2 transcripts but not the mMCP-4, mMCP-5, mMCP-6, or mMCP-7 transcripts. Although these two histochemically distinct populations of tissue mast cells apparently express their granule proteases in a strict subclass-specific manner, nontransformed but immature populations of BALB/cJ mouse mast cells derived in vitro can express varied combinations of the eight mMCPs in response to different recombinant cytokines (12)(13)(14)(15)(16).In addition to those protease transcripts expressed by SMCs. We also present RNA and immunohistochemical data that indicate that the WBB6F1/J-+/+, WCB6F1/J-+/+, WB/ ReJ-+/+, and WC/ReJ-+/+ strains of mice differ from the BALB/cJ mouse strain in their prominent expression of mMCP-2 in both their SMCs and perivascular CTMCs. MATERIALS AND METHODSMouse Mast-Cell-Specific Protease mRNA Levels in Ear and Skin CTMCs and Isolated SMCs. Female mice of the WB/ ReJ-+/+, WC/ReJ-+/+, WBB6F1/J-+/+, WCB6F1/J-+/+, WBB6F,/J-W/Wv, WCB...
SummaryThe ear, skin, and purified serosal mast calls of WBB6F1/J-+/+ (WB-+/+) and WCB6Ft/J-+/+ (WC-+/+) mice contain high steady-state levels of the transcripts that encode mouse mast cell protease (mMCP) 2, mMCP-4, mMCP-5, mMCP-6, and mouse mast cell carboxypeptidase A (mMC-CPA). In contrast, no mast cell protease transcripts are present in abundance in the ear and skin of and WCB6F1/J-SI/S1 d (SI/SI a) mice which are mast cell-deficient in vivo due to defects in their c-kit and c-kit ligand genes, respectively. We now report that the immature bone marrow-derived mast cells (mBMMC) obtained in vitro with recombinant interleukin 3 (rIL-3) or WEHI-3 cell conditioned medium from WB-+/+, WC-+/+, W/W ~, and SI/SI d mice all contain high steady-state levels of the mMCP-2, mMCP-4, mMCP-5, mMCP-6, and mMC-CPA transcripts. As assessed immunohistochemically, mMCP-2 protein and mMCP-5 protein are also present in the granules of mBMMC from WB-+/+, WC-+/+, and W/W ~ mice. That SI/SI d and W/W ~ mBMMC contain high steady-state levels of five granule protease transcripts expressed by the mature serosal, ear, and skin mast cells of their normal +/+ littermates suggests that c-kit-mediated signal transduction is not essential for inducing transcription of these protease genes. Because rlL-4 inhibits the rlL-10-induced expression of mMCP-1 and mMCP-2 in BALB/cJ mBMMC, the ability of rlL-4 to influence protease mRNA levels in WC-+/+ mBMMC and W/W ~ mBMMC was investigated. Although rlL-10 induced expression of the mMCP-1 transcript in WC-+/+ and W/W ~ mBMMC, rIL-4 was not able to suppress the steady-state levels of the mMCP-1 transcript or any other protease transcript in these cuhured mast cells. Thus, not only do BALB/cJ mBMMC express fewer granule proteases than mBMMC from mast cell-deficient strains and their normal littermates but the subsequent induction of late-expressed proteases in BALB/cJ mBMMC is more tightly regulated by IL-3 and IL-4.
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