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The chymase, mouse mast cell protease 4, constitutes the major chymotrypsin-like activity in peritoneum and ear tissue. A role for mouse mast cell protease 4 in thrombin regulation and fibronectin turnover.

Tchougounova E, Pejler G, Abrink M - J. Exp. Med. (2003)

Bottom Line: However, mMCP-4 inactivation resulted in complete loss of chymotryptic activity in the peritoneum and in ear tissue, indicating that mMCP-4 is the main source of stored chymotrypsin-like protease activity at these sites.The mMCP-4 cells showed markedly impaired ability to perform inactivating cleavages of thrombin, indicating a role for mMCP-4 in regulating the extravascular coagulation system.Further, a role for mMCP-4 in connective tissue remodeling was suggested by the inability of mMCP-4 peritoneal cells to process endogenous fibronectin.

View Article: PubMed Central - PubMed

Affiliation: Swedish University of Agricultural Sciences, Department of Molecular Biosciences, The Biomedical Center, Box 575, 751 23 Uppsala, Sweden.

ABSTRACT
To gain insight into the biological role of mast cell chymase we have generated a mouse strain with a targeted deletion in the gene for mast cell protease 4 (mMCP-4), the mouse chymase that has the closest relationship to the human chymase in terms of tissue localization and functional properties. The inactivation of mMCP-4 did not affect the storage of other mast cell proteases and did not affect the number of mast cells or the mast cell morphology. However, mMCP-4 inactivation resulted in complete loss of chymotryptic activity in the peritoneum and in ear tissue, indicating that mMCP-4 is the main source of stored chymotrypsin-like protease activity at these sites. The mMCP-4 cells showed markedly impaired ability to perform inactivating cleavages of thrombin, indicating a role for mMCP-4 in regulating the extravascular coagulation system. Further, a role for mMCP-4 in connective tissue remodeling was suggested by the inability of mMCP-4 peritoneal cells to process endogenous fibronectin.

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(A) Genomic organization of the mouse MC chymase locus. The positions of the different chymase genes and their transcriptional orientations are indicated by arrows. mMCP-5 and mMCP-8, which are clearly different from the other chymases in terms of structure or tissue specificity (see Results), are positioned at the flanks of the locus. The more closely related chymases mMCP-1, -2, -4, and –9 are clustered in the middle of the locus. (B) Construct used for targeting of the mMCP-4 gene. Detailed organization of a 16 kb genomic fragment covering the mMCP-4 gene and 12 kb of upstream regions. EcoRI, BglII, and BamHI indicate restriction sites found. Depicted is also the final targeting construct used for homologous recombination. Neo corresponds to the neomycin resistant cassette used in the target vector, with promotor (PGK) and transcriptional orientation (arrow) indicated. Roman numerals indicate the positions of primers used in genomic PCR (primer I is upstream outside construct, II is inside Neo, III is in exon 1, IV is in exon 2, V is reverse complementary to II, VI is downstream outside construct). (C) PCR screening of targeted mice. Genomic DNA was prepared from WT (+/+), heterozygous (+/−) or homozygous (−/−) littermates and was subjected to PCR analysis to trace the mMCP-4 mutation. PCR products were separated on 1% agarose gels. The top panel shows a standard result using primers I+II, yielding the 5 kb product characteristic of the mutant mMCP-4 gene. The bottom panel shows the result obtained using primers I+IV, where the insertion of the mutant mMCP-4 gene results in a 7 kb PCR product.
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fig1: (A) Genomic organization of the mouse MC chymase locus. The positions of the different chymase genes and their transcriptional orientations are indicated by arrows. mMCP-5 and mMCP-8, which are clearly different from the other chymases in terms of structure or tissue specificity (see Results), are positioned at the flanks of the locus. The more closely related chymases mMCP-1, -2, -4, and –9 are clustered in the middle of the locus. (B) Construct used for targeting of the mMCP-4 gene. Detailed organization of a 16 kb genomic fragment covering the mMCP-4 gene and 12 kb of upstream regions. EcoRI, BglII, and BamHI indicate restriction sites found. Depicted is also the final targeting construct used for homologous recombination. Neo corresponds to the neomycin resistant cassette used in the target vector, with promotor (PGK) and transcriptional orientation (arrow) indicated. Roman numerals indicate the positions of primers used in genomic PCR (primer I is upstream outside construct, II is inside Neo, III is in exon 1, IV is in exon 2, V is reverse complementary to II, VI is downstream outside construct). (C) PCR screening of targeted mice. Genomic DNA was prepared from WT (+/+), heterozygous (+/−) or homozygous (−/−) littermates and was subjected to PCR analysis to trace the mMCP-4 mutation. PCR products were separated on 1% agarose gels. The top panel shows a standard result using primers I+II, yielding the 5 kb product characteristic of the mutant mMCP-4 gene. The bottom panel shows the result obtained using primers I+IV, where the insertion of the mutant mMCP-4 gene results in a 7 kb PCR product.

Mentions: A ∼16 kb fragment of the mMCP-4 gene was cloned from a 129SVJ mouse genomic lambda FIX II library (Stratagene) using a full-length mMCP-4 cDNA as probe (a kind gift from Lars Hellman [Uppsala University, Sweden]). The 16 kb genomic fragment was released from the library vector with NotI enzyme and subcloned into the pZero vector (Invitrogen). The subcloning allowed further characterization of the structure of the mMCP-4 gene and identification of suitable restriction sites for construction of the target vector to be used for homologous recombination in ES cells. A BamH1 site was identified between exons 1 and 2, and this site together with the NotI site of the pZero/lamda vector was used for cloning of a ∼4.5 kb downstream arm of the mMCP-4 gene into the target vector (Fig. 1 B). A suitable ∼3 kb fragment with EcoRI and BglII sites was found upstream of the mMCP-4 gene, covering the promotor region (Fig. 1 B). The blunted EcoRI and BglII sites were used for cloning of the upstream arm into a blunted XhoI site of the target vector containing the neo cassette (indicated by solid lines connecting the upper and lower parts of Fig. 1 B). This strategy thus deletes exon 1 of the mMCP-4 gene. The targeting vector was electroporated into ES cells using 1.5 μg of DNA/106 ES cells. The ES cell work was performed using a standard protocol but without negative selection, i.e., omitting the selection against thymidine kinase expression. Cell cultures derived from the different ES cell clones obtained were divided into two portions. One of the portions was frozen (−80°C) whereas the remaining cells were cultured further and used for preparation of genomic DNA.


The chymase, mouse mast cell protease 4, constitutes the major chymotrypsin-like activity in peritoneum and ear tissue. A role for mouse mast cell protease 4 in thrombin regulation and fibronectin turnover.

Tchougounova E, Pejler G, Abrink M - J. Exp. Med. (2003)

(A) Genomic organization of the mouse MC chymase locus. The positions of the different chymase genes and their transcriptional orientations are indicated by arrows. mMCP-5 and mMCP-8, which are clearly different from the other chymases in terms of structure or tissue specificity (see Results), are positioned at the flanks of the locus. The more closely related chymases mMCP-1, -2, -4, and –9 are clustered in the middle of the locus. (B) Construct used for targeting of the mMCP-4 gene. Detailed organization of a 16 kb genomic fragment covering the mMCP-4 gene and 12 kb of upstream regions. EcoRI, BglII, and BamHI indicate restriction sites found. Depicted is also the final targeting construct used for homologous recombination. Neo corresponds to the neomycin resistant cassette used in the target vector, with promotor (PGK) and transcriptional orientation (arrow) indicated. Roman numerals indicate the positions of primers used in genomic PCR (primer I is upstream outside construct, II is inside Neo, III is in exon 1, IV is in exon 2, V is reverse complementary to II, VI is downstream outside construct). (C) PCR screening of targeted mice. Genomic DNA was prepared from WT (+/+), heterozygous (+/−) or homozygous (−/−) littermates and was subjected to PCR analysis to trace the mMCP-4 mutation. PCR products were separated on 1% agarose gels. The top panel shows a standard result using primers I+II, yielding the 5 kb product characteristic of the mutant mMCP-4 gene. The bottom panel shows the result obtained using primers I+IV, where the insertion of the mutant mMCP-4 gene results in a 7 kb PCR product.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2194091&req=5

fig1: (A) Genomic organization of the mouse MC chymase locus. The positions of the different chymase genes and their transcriptional orientations are indicated by arrows. mMCP-5 and mMCP-8, which are clearly different from the other chymases in terms of structure or tissue specificity (see Results), are positioned at the flanks of the locus. The more closely related chymases mMCP-1, -2, -4, and –9 are clustered in the middle of the locus. (B) Construct used for targeting of the mMCP-4 gene. Detailed organization of a 16 kb genomic fragment covering the mMCP-4 gene and 12 kb of upstream regions. EcoRI, BglII, and BamHI indicate restriction sites found. Depicted is also the final targeting construct used for homologous recombination. Neo corresponds to the neomycin resistant cassette used in the target vector, with promotor (PGK) and transcriptional orientation (arrow) indicated. Roman numerals indicate the positions of primers used in genomic PCR (primer I is upstream outside construct, II is inside Neo, III is in exon 1, IV is in exon 2, V is reverse complementary to II, VI is downstream outside construct). (C) PCR screening of targeted mice. Genomic DNA was prepared from WT (+/+), heterozygous (+/−) or homozygous (−/−) littermates and was subjected to PCR analysis to trace the mMCP-4 mutation. PCR products were separated on 1% agarose gels. The top panel shows a standard result using primers I+II, yielding the 5 kb product characteristic of the mutant mMCP-4 gene. The bottom panel shows the result obtained using primers I+IV, where the insertion of the mutant mMCP-4 gene results in a 7 kb PCR product.
Mentions: A ∼16 kb fragment of the mMCP-4 gene was cloned from a 129SVJ mouse genomic lambda FIX II library (Stratagene) using a full-length mMCP-4 cDNA as probe (a kind gift from Lars Hellman [Uppsala University, Sweden]). The 16 kb genomic fragment was released from the library vector with NotI enzyme and subcloned into the pZero vector (Invitrogen). The subcloning allowed further characterization of the structure of the mMCP-4 gene and identification of suitable restriction sites for construction of the target vector to be used for homologous recombination in ES cells. A BamH1 site was identified between exons 1 and 2, and this site together with the NotI site of the pZero/lamda vector was used for cloning of a ∼4.5 kb downstream arm of the mMCP-4 gene into the target vector (Fig. 1 B). A suitable ∼3 kb fragment with EcoRI and BglII sites was found upstream of the mMCP-4 gene, covering the promotor region (Fig. 1 B). The blunted EcoRI and BglII sites were used for cloning of the upstream arm into a blunted XhoI site of the target vector containing the neo cassette (indicated by solid lines connecting the upper and lower parts of Fig. 1 B). This strategy thus deletes exon 1 of the mMCP-4 gene. The targeting vector was electroporated into ES cells using 1.5 μg of DNA/106 ES cells. The ES cell work was performed using a standard protocol but without negative selection, i.e., omitting the selection against thymidine kinase expression. Cell cultures derived from the different ES cell clones obtained were divided into two portions. One of the portions was frozen (−80°C) whereas the remaining cells were cultured further and used for preparation of genomic DNA.

Bottom Line: However, mMCP-4 inactivation resulted in complete loss of chymotryptic activity in the peritoneum and in ear tissue, indicating that mMCP-4 is the main source of stored chymotrypsin-like protease activity at these sites.The mMCP-4 cells showed markedly impaired ability to perform inactivating cleavages of thrombin, indicating a role for mMCP-4 in regulating the extravascular coagulation system.Further, a role for mMCP-4 in connective tissue remodeling was suggested by the inability of mMCP-4 peritoneal cells to process endogenous fibronectin.

View Article: PubMed Central - PubMed

Affiliation: Swedish University of Agricultural Sciences, Department of Molecular Biosciences, The Biomedical Center, Box 575, 751 23 Uppsala, Sweden.

ABSTRACT
To gain insight into the biological role of mast cell chymase we have generated a mouse strain with a targeted deletion in the gene for mast cell protease 4 (mMCP-4), the mouse chymase that has the closest relationship to the human chymase in terms of tissue localization and functional properties. The inactivation of mMCP-4 did not affect the storage of other mast cell proteases and did not affect the number of mast cells or the mast cell morphology. However, mMCP-4 inactivation resulted in complete loss of chymotryptic activity in the peritoneum and in ear tissue, indicating that mMCP-4 is the main source of stored chymotrypsin-like protease activity at these sites. The mMCP-4 cells showed markedly impaired ability to perform inactivating cleavages of thrombin, indicating a role for mMCP-4 in regulating the extravascular coagulation system. Further, a role for mMCP-4 in connective tissue remodeling was suggested by the inability of mMCP-4 peritoneal cells to process endogenous fibronectin.

Show MeSH
Related in: MedlinePlus