C/EBP in Asthma
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《新英格兰医药杂志》
To the Editor: Roth et al. (Aug. 5 issue)1 report that glucocorticoids do not inhibit the proliferation of bronchial smooth-muscle cells from patients with asthma and propose that these cells lack expression of the CCAAT/enhancer binding protein (C/EBP), a transcription factor. However, Figure 4A in their article shows reactivity of an anti-C/EBP antibody to a protein band about 4 kD below what is seen for patients with emphysema. This raises the possibility that a different isoform of C/EBP is expressed, possibly as a result of alternate RNA splicing, aberrant glycosylation, or up-regulation of a cleavage enzyme of C/EBP. The dual-banding pattern seen when the bronchial smooth-muscle cells from patients with asthma were transiently transfected with C/EBP (Figure 4C) suggests that a proteolytic enzyme is active.
Is it possible that the bronchial smooth-muscle cells of patients with asthma actually do express C/EBP, but that the glucocorticoid-receptor-–binding region is cleaved off? Such a possibility has important mechanistic implications.
Daniel I. Levy, M.D., Ph.D.
University of Chicago School of Medicine
Chicago, IL 60637
dlevy@medicine.bsd.uchicago.edu
References
Roth M, Johnson PR, Borger P, et al. Dysfunctional interaction of C/EBP and the glucocorticoid receptor in asthmatic bronchial smooth-muscle cells. N Engl J Med 2004;351:560-574.
The authors reply: We agree with Dr. Levy that the expression of small C/EBP bands (Figure 4A of our article) indicates the presence of small isoforms of C/EBP in a subgroup of patients with asthma. It has to be emphasized that the full-length C/EBP protein is missing in all 20 asthmatic cell lines. The expression of C/EBP isoforms is not regulated by differential splicing but depends on alternative translation-initiation sites in C/EBP messenger RNA.1,2 Four alternative translation-initiation sites have been described in human C/EBP.1 The dual bands observed in bronchial smooth-muscle cells from patients with asthma after transfection with C/EBP may be explained by these different translation-initiation sites, and transfected genes are not naturally processed. We agree that cleaving of the glucocorticoid-binding region would abrogate binding of C/EBP to the glucocorticoid receptor and to its DNA consensus sequence.3,4 The antibody used in our experiments detects all known C/EBP isoforms; therefore, it is unlikely that we missed C/EBP variants in asthmatic cells.
Michael Roth, Ph.D.
Peter Borger, Ph.D.
Michael Tamm, M.D.
University Hospital Basel
CH-4031 Basel, Switzerland
michaelr@med.usyd.edu.au
References
Calkhoven CF, Bouwman PR, Snippe L, Ab G. Translation start multiplicity of the CCAAT/enhancer binding protein alpha mRNA is dictated by a small 5' open reading frame. Nucleic Acids Res 1999;22:554-557.
Calkhoven CF, Muller C, Leutz A. Translational control of C/EBPalpha and C/EBPbeta isoform expression. Genes Dev 2000;14:1920-1932.
Ramos RA, Meilandt WJ, Wang EC, Firestone GL. Dysfunctional glucocorticoid receptor with a single point mutation ablates the CCAAT/enhancer binding protein-dependent growth suppression response in a steroid-resistant rat hepatoma cell variant. FASEB J 1999;13:169-180.
Roth M, Johnson PRA, Rüdiger JJ, et al. Interaction between glucocorticoids and 2 agonists on bronchial airway smooth muscle cells through synchronised cellular signalling. Lancet 2002;360:1293-1299.
Is it possible that the bronchial smooth-muscle cells of patients with asthma actually do express C/EBP, but that the glucocorticoid-receptor-–binding region is cleaved off? Such a possibility has important mechanistic implications.
Daniel I. Levy, M.D., Ph.D.
University of Chicago School of Medicine
Chicago, IL 60637
dlevy@medicine.bsd.uchicago.edu
References
Roth M, Johnson PR, Borger P, et al. Dysfunctional interaction of C/EBP and the glucocorticoid receptor in asthmatic bronchial smooth-muscle cells. N Engl J Med 2004;351:560-574.
The authors reply: We agree with Dr. Levy that the expression of small C/EBP bands (Figure 4A of our article) indicates the presence of small isoforms of C/EBP in a subgroup of patients with asthma. It has to be emphasized that the full-length C/EBP protein is missing in all 20 asthmatic cell lines. The expression of C/EBP isoforms is not regulated by differential splicing but depends on alternative translation-initiation sites in C/EBP messenger RNA.1,2 Four alternative translation-initiation sites have been described in human C/EBP.1 The dual bands observed in bronchial smooth-muscle cells from patients with asthma after transfection with C/EBP may be explained by these different translation-initiation sites, and transfected genes are not naturally processed. We agree that cleaving of the glucocorticoid-binding region would abrogate binding of C/EBP to the glucocorticoid receptor and to its DNA consensus sequence.3,4 The antibody used in our experiments detects all known C/EBP isoforms; therefore, it is unlikely that we missed C/EBP variants in asthmatic cells.
Michael Roth, Ph.D.
Peter Borger, Ph.D.
Michael Tamm, M.D.
University Hospital Basel
CH-4031 Basel, Switzerland
michaelr@med.usyd.edu.au
References
Calkhoven CF, Bouwman PR, Snippe L, Ab G. Translation start multiplicity of the CCAAT/enhancer binding protein alpha mRNA is dictated by a small 5' open reading frame. Nucleic Acids Res 1999;22:554-557.
Calkhoven CF, Muller C, Leutz A. Translational control of C/EBPalpha and C/EBPbeta isoform expression. Genes Dev 2000;14:1920-1932.
Ramos RA, Meilandt WJ, Wang EC, Firestone GL. Dysfunctional glucocorticoid receptor with a single point mutation ablates the CCAAT/enhancer binding protein-dependent growth suppression response in a steroid-resistant rat hepatoma cell variant. FASEB J 1999;13:169-180.
Roth M, Johnson PRA, Rüdiger JJ, et al. Interaction between glucocorticoids and 2 agonists on bronchial airway smooth muscle cells through synchronised cellular signalling. Lancet 2002;360:1293-1299.