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Role of P-selectin cytoplasmic domain in granular targeting in vivo and in early inflammatory responses.

Hartwell DW, Mayadas TN, Berger G, Frenette PS, Rayburn H, Hynes RO, Wagner DD - J. Cell Biol. (1998)

Bottom Line: The deletion did not affect the sorting of P-selectin into alpha-granules of platelets but severely compromised the storage of P-selectin in endothelial cells.Unstored P-selectin was proteolytically shed from the plasma membrane, resulting in increased levels of soluble P-selectin in the plasma.Our results suggest that different sorting mechanisms for P-selectin are used in platelets and endothelial cells and that the storage pool of P-selectin in endothelial cells is functionally important during early stages of inflammation.

View Article: PubMed Central - PubMed

Affiliation: Center for Blood Research, Harvard Medical School, Boston, Massachusetts 02115, USA.

ABSTRACT
P-selectin is an adhesion receptor for leukocytes expressed on activated platelets and endothelial cells. The cytoplasmic domain of P-selectin was shown in vitro to contain signals required for both the sorting of this protein into storage granules and its internalization from the plasma membrane. To evaluate in vivo the role of the regulated secretion of P-selectin, we have generated a mouse that expresses P-selectin lacking the cytoplasmic domain (DeltaCT mice). The deletion did not affect the sorting of P-selectin into alpha-granules of platelets but severely compromised the storage of P-selectin in endothelial cells. Unstored P-selectin was proteolytically shed from the plasma membrane, resulting in increased levels of soluble P-selectin in the plasma. The DeltaCT-P-selectin appeared capable of mediating cell adhesion as it supported leukocyte rolling in the mutant mice. However, a secretagogue failed to upregulate leukocyte rolling in the DeltaCT mice, indicating an absence of a releasable storage pool of P-selectin in the endothelium. Furthermore, the neutrophil influx into the inflamed peritoneum was only 30% of the wild-type level 2 h after stimulation. Our results suggest that different sorting mechanisms for P-selectin are used in platelets and endothelial cells and that the storage pool of P-selectin in endothelial cells is functionally important during early stages of inflammation.

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Northern blot analysis of RNA samples (25 μg/ lane) from lung tissues of  mice treated with LPS for 3.5  h. Wild-type (wt), heterozygous (het), and homozygous  (homo) mutants are as indicated. The cDNA probe is a  1.6-kb fragment from the 3′  half of P-selectin. TM and  CT probes are 66-base and  72-base antisense oligonucleotides of the respective domains. The hGH probe is a  63-base oligonucleotide from  hGH 3′-UTR. The diagram  depicts the predicted wild-type and mutant mRNA. Asterisk, the aberrant minor  mRNA species discussed in  the text.
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Figure 2: Northern blot analysis of RNA samples (25 μg/ lane) from lung tissues of mice treated with LPS for 3.5 h. Wild-type (wt), heterozygous (het), and homozygous (homo) mutants are as indicated. The cDNA probe is a 1.6-kb fragment from the 3′ half of P-selectin. TM and CT probes are 66-base and 72-base antisense oligonucleotides of the respective domains. The hGH probe is a 63-base oligonucleotide from hGH 3′-UTR. The diagram depicts the predicted wild-type and mutant mRNA. Asterisk, the aberrant minor mRNA species discussed in the text.

Mentions: To determine whether the mutated P-selectin gene was expressed in homozygous mutant mice, total RNA was extracted from the lungs of wild-type, heterozygous, and homozygous animals after treatment with lipopolysaccharide (LPS) for 3.5 h to stimulate P-selectin synthesis (Sanders et al., 1992). A Northern blot probed with murine P-selectin cDNA revealed a 3-kb transcript in wild-type mice as expected (Fig. 2). A truncated transcript of 2.2 kb encoding domains from the NH2-terminal to the transmembrane domain with the addition of the hGH 3′-UTR was predicted to be expressed by heterozygous as well as the homozygous mice. As shown in Fig. 2, indeed both the wild-type and the truncated transcripts were present in heterozygous mice. In homozygous mice, the truncated mRNA species represented the majority of the message detected by the P-selectin cDNA probe. However, the P-selectin cDNA probe also detected two minor bands at higher molecular weight (Fig. 2, asterisk) in homozygous mutants. Subsequent probing with oligo antisense DNA probes revealed that these upper bands contained sequences of the transmembrane and cytoplasmic domain, and the 3′-UTR of the hGH gene. These results show that some read-through transcription beyond the hGH transcription terminator must occur at a reduced level to include sequences from the neomycin cassette and C1 and C2 exon. These transcripts could then be spliced to produce mRNAs which include sequences of C1 and C2 exons. Although the exact splicing events cannot readily be determined, one can deduce the following possible outcomes. First, any mRNA that correctly splices exon CR8 and exon TM will necessarily terminate translation at the termination codons introduced during the mutagenesis, whatever other sequences follow in the mRNA. All such mRNAs will therefore encode the truncated membrane-bound form without CT as anticipated. Any aberrant splicing events that exclude the TM will yield secreted proteins. The most readily conceived would be an aberrant splice from CR8 to C1. If such a splice were to occur, the reading frame would be preserved so that any resulting protein would react with the antibody against the CT. The same would be the case if any other CR exon were to splice to C1 since all CR exons are in the same phase (Johnston et al., 1990; Sanders et al., 1992). As will be discussed below, no protein containing the P-selectin CT was found in the ΔCT mice.


Role of P-selectin cytoplasmic domain in granular targeting in vivo and in early inflammatory responses.

Hartwell DW, Mayadas TN, Berger G, Frenette PS, Rayburn H, Hynes RO, Wagner DD - J. Cell Biol. (1998)

Northern blot analysis of RNA samples (25 μg/ lane) from lung tissues of  mice treated with LPS for 3.5  h. Wild-type (wt), heterozygous (het), and homozygous  (homo) mutants are as indicated. The cDNA probe is a  1.6-kb fragment from the 3′  half of P-selectin. TM and  CT probes are 66-base and  72-base antisense oligonucleotides of the respective domains. The hGH probe is a  63-base oligonucleotide from  hGH 3′-UTR. The diagram  depicts the predicted wild-type and mutant mRNA. Asterisk, the aberrant minor  mRNA species discussed in  the text.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132959&req=5

Figure 2: Northern blot analysis of RNA samples (25 μg/ lane) from lung tissues of mice treated with LPS for 3.5 h. Wild-type (wt), heterozygous (het), and homozygous (homo) mutants are as indicated. The cDNA probe is a 1.6-kb fragment from the 3′ half of P-selectin. TM and CT probes are 66-base and 72-base antisense oligonucleotides of the respective domains. The hGH probe is a 63-base oligonucleotide from hGH 3′-UTR. The diagram depicts the predicted wild-type and mutant mRNA. Asterisk, the aberrant minor mRNA species discussed in the text.
Mentions: To determine whether the mutated P-selectin gene was expressed in homozygous mutant mice, total RNA was extracted from the lungs of wild-type, heterozygous, and homozygous animals after treatment with lipopolysaccharide (LPS) for 3.5 h to stimulate P-selectin synthesis (Sanders et al., 1992). A Northern blot probed with murine P-selectin cDNA revealed a 3-kb transcript in wild-type mice as expected (Fig. 2). A truncated transcript of 2.2 kb encoding domains from the NH2-terminal to the transmembrane domain with the addition of the hGH 3′-UTR was predicted to be expressed by heterozygous as well as the homozygous mice. As shown in Fig. 2, indeed both the wild-type and the truncated transcripts were present in heterozygous mice. In homozygous mice, the truncated mRNA species represented the majority of the message detected by the P-selectin cDNA probe. However, the P-selectin cDNA probe also detected two minor bands at higher molecular weight (Fig. 2, asterisk) in homozygous mutants. Subsequent probing with oligo antisense DNA probes revealed that these upper bands contained sequences of the transmembrane and cytoplasmic domain, and the 3′-UTR of the hGH gene. These results show that some read-through transcription beyond the hGH transcription terminator must occur at a reduced level to include sequences from the neomycin cassette and C1 and C2 exon. These transcripts could then be spliced to produce mRNAs which include sequences of C1 and C2 exons. Although the exact splicing events cannot readily be determined, one can deduce the following possible outcomes. First, any mRNA that correctly splices exon CR8 and exon TM will necessarily terminate translation at the termination codons introduced during the mutagenesis, whatever other sequences follow in the mRNA. All such mRNAs will therefore encode the truncated membrane-bound form without CT as anticipated. Any aberrant splicing events that exclude the TM will yield secreted proteins. The most readily conceived would be an aberrant splice from CR8 to C1. If such a splice were to occur, the reading frame would be preserved so that any resulting protein would react with the antibody against the CT. The same would be the case if any other CR exon were to splice to C1 since all CR exons are in the same phase (Johnston et al., 1990; Sanders et al., 1992). As will be discussed below, no protein containing the P-selectin CT was found in the ΔCT mice.

Bottom Line: The deletion did not affect the sorting of P-selectin into alpha-granules of platelets but severely compromised the storage of P-selectin in endothelial cells.Unstored P-selectin was proteolytically shed from the plasma membrane, resulting in increased levels of soluble P-selectin in the plasma.Our results suggest that different sorting mechanisms for P-selectin are used in platelets and endothelial cells and that the storage pool of P-selectin in endothelial cells is functionally important during early stages of inflammation.

View Article: PubMed Central - PubMed

Affiliation: Center for Blood Research, Harvard Medical School, Boston, Massachusetts 02115, USA.

ABSTRACT
P-selectin is an adhesion receptor for leukocytes expressed on activated platelets and endothelial cells. The cytoplasmic domain of P-selectin was shown in vitro to contain signals required for both the sorting of this protein into storage granules and its internalization from the plasma membrane. To evaluate in vivo the role of the regulated secretion of P-selectin, we have generated a mouse that expresses P-selectin lacking the cytoplasmic domain (DeltaCT mice). The deletion did not affect the sorting of P-selectin into alpha-granules of platelets but severely compromised the storage of P-selectin in endothelial cells. Unstored P-selectin was proteolytically shed from the plasma membrane, resulting in increased levels of soluble P-selectin in the plasma. The DeltaCT-P-selectin appeared capable of mediating cell adhesion as it supported leukocyte rolling in the mutant mice. However, a secretagogue failed to upregulate leukocyte rolling in the DeltaCT mice, indicating an absence of a releasable storage pool of P-selectin in the endothelium. Furthermore, the neutrophil influx into the inflamed peritoneum was only 30% of the wild-type level 2 h after stimulation. Our results suggest that different sorting mechanisms for P-selectin are used in platelets and endothelial cells and that the storage pool of P-selectin in endothelial cells is functionally important during early stages of inflammation.

Show MeSH
Related in: MedlinePlus