Angiotensin converting enzyme 2 is primarily epithelial and is developmentally regulated in the mouse lung

Abstract

Angiotensin converting enzyme (ACE) 2 is a carboxypeptidase that shares 42% amino acid homology with ACE. Little is known about the regulation or pattern of expression of ACE2 in the mouse lung, including its definitive cellular distribution or developmental changes. Based on Northern blot and RT-PCR data, we report two distinct transcripts of ACE2 in the mouse lung and kidney and describe a 5' exon 1a previously unidentified in the mouse. Western blots show multiple isoforms of ACE2, with predominance of a 75-80 kDa protein in the mouse lung versus a 120 kDa form in the mouse kidney. Immunohistochemistry localizes ACE2 protein to Clara cells, type II cells, and endothelium and smooth muscle of small and medium vessels in the mouse lung. ACE2 mRNA levels peak at embryonic day 18.5 in the mouse lung, and immunostaining demonstrates protein primarily in the bronchiolar epithelium at that developmental time point. In murine cell lines ACE2 is strongly expressed in the Clara cell line mtCC, as opposed to the low mRNA expression detected in E10 (type I-like alveolar epithelial cell line), MLE-15 (type II alveolar epithelial cell line), MFLM-4 (fetal pulmonary vasculature cell line), and BUMPT-7 (renal proximal tubule cell line). In summary, murine pulmonary ACE2 appears to be primarily epithelial, is developmentally regulated, and has two transcripts that include a previously undescribed exon.

Figure 1

Identification of murine exon 1a. A: Northern blot for ACE2 expression in murine tissues and cell lines. The left panel represents Northern blot developed after 7 days exposure, the right panel shows blot developed after 10 days exposure. Lane m: 1 kb DNA marker (indicated in kb pairs); L: adult lung; K: adult kidney; cc: mtCC cells. B: RT‐PCR for putative 5′ exon 1a of murine ACE2. Lane m: 100 bp DNA marker (indicated in kb pairs); w: water; GD: genomic DNA; L: adult lung; K: adult kidney; cc: mtCC cells. C: 5′‐RACE for putative exon 1a in adult lung. By sequencing ∼800 bp band represents non‐specific amplification and ∼300 bp band includes the 188 bp exon 1a of murine ACE2. Lane m: 100 bp DNA marker; L: adult lung. D: Sequence of human (H) and mouse (M) exon 1a. Capital letters: identitical nucleotides in both species; lower case letters: divergent nucleotides in mouse and human; hyphen (‐): gap in sequence. Italics indicate bordering intron sequence in mouse genome.

Figure 2

Transcripts of murine ACE2. A: Schematic showing splice products of published ACE2 mRNA and novel exon 1a into possible transcripts. Arrows indicate forward and reverse primers used for RT‐PCR. B: RT‐PCR for ACE2 transcripts in adult lung. m: 500 bp marker (indicated in kb pairs); 1–4: transcripts 1–4 as shown in (A), with transcript 1 amplified using forward primer 1a‐F and reverse primer BC‐R, transcript 2 using forward primer 1a‐F and reverse primer NM‐R, transcript 3 using forward primer BC‐F and reverse primer BC‐R, transcript 4 using forward primer BC‐F and reverse primer NM‐R. C: RT‐PCR showing presence of ACE2 transcript 2 (amplified using forward primer 1a‐F and reverse primer NM‐R), as shown in (A) in various murine tissues and cell lines. w: water; K: adult kidney; H: adult heart; T: adult testis; L: adult lung; 18L: embryonic day 18.5 lung; cc: mtCC cells.

Figure 3

Multiple protein species specific for ACE2. A: Western blot for ACE2 protein in murine tissues and cell lines using anti‐ACE2 Abcam ab15348 against cytoplasmic C‐terminus. Lane m: p7708s protein marker (in kDa); L: adult lung; K: adult kidney; H: adult heart; T: adult testis; cc: mtCC; e10: E10; mle: MLE‐15; mflm: MFLM‐4; pt: BUMPT‐7. B: Western blot for ACE2 protein in murine tissues and cell lines using anti‐ACE2 R&D Systems AF933 against N‐terminal ectodomain. Lane m: p7708s protein marker (in kDa); cc: mtCC; L: adult lung; K: adult kidney. C: Western blot for ACE2 protein using anti‐ACE2 Santa Cruz sc21834 alone. Lane m: p7708s protein marker (in kDa); e10: E10; L: adult lung; K: adult kidney. D: Competitive inhibition Western blot for ACE2 protein using anti‐ACE2 Santa Cruz sc21834 in the presence of specific ACE2 control peptide sc21834‐P. Lane m: p7708s protein marker (in kDa); e10: E10; L: adult lung; K: adult kidney. E: Competitive inhibition Western blot for ACE2 protein using anti‐ACE2 Santa Cruz sc21834 in the presence of non‐specific tuberin control peptide sc893‐P. Lane m: p7708s protein marker (in kDa); e10: E10; L: adult lung; K: adult kidney.

Figure 4

ACE2 expression in murine tissues and cell lines. A: RT‐PCR for ACE2 and β‐actin expression in murine tissues and cell lines (40 cycles). Lane w: water; L: adult lung; H: adult heart; K: adult kidney; pt: BUMPT‐7; e: E10; le: MLE‐15; cc: mtCC; mf: MFLM‐4. B: Nested RT‐PCR for ACE2 expression in murine tissues and cell lines. Lane w: water; L: adult lung; K: adult kidney; H: adult heart; T: adult testis; cc: mtCC; e: E10; le: MLE‐15; mf: MFLM‐4; pt: BUMPT‐7. C: QRT‐PCR for ACE2 expression in murine cell lines, normalized to 18S. cc: mtCC; mle: MLE‐15; mflm: MFLM‐4; pt: BUMPT‐7. *P<0.005 compared to other cell lines.

Figure 5

Immunohistochemistry for ACE2 in murine tissues. Red arrow: positive staining for ACE2; yellow arrow: no staining for ACE2. Br: bronchiole; BV: blood vessel; A: alveolar space; M: medulla; C: cortex; PT: proximal tubule. A: Adult lung. Red arrow: bronchiolar epithelium. B: Adult lung. Red arrow: Clara cell; yellow arrow: arteriolar endothelium. C: Adult lung. Red arrow: Clara cell; yellow arrow: ciliated epithelium. D: Adult lung. Red arrow: Clara cell; yellow arrow: ciliated epithelium. E: Adult lung. Red arrow: vascular endothelium. F–H: Adult lung type II alveolar epithelial cells. I: Adult lung. Red arrow: endothelium of venule. J: E18.5 lung. Red arrow: bronchiolar epithelium. K: E18.5 lung. Red arrow: Clara cell; yellow arrow: ciliated epithelium. L,M: Adult kidney. Cortex stains positive for ACE2; medulla is negative for ACE2. N,O: Adult kidney. Red arrow: proximal tubule brush border. P: Adult kidney. Red arrow: endothelium and smooth muscle of small artery. Q: Adult myocardium. Red arrows: capillary endothelium. Panels A–C,F–H,L,N,P: antibody sc21834; other panels ab15348. Glutaraldehyde (0.1%) was added to fixative for panels A–C,F–K,Q. Panels L,M,O,P are frozen rather than paraffin‐embedded tissues. Antigen retrieval was performed on tissues in panels A–C,F–K,N,Q.

Figure 6

Developmental pattern of ACE2 expression. A: RT‐PCR for ACE2, β‐actin, T1α, and caveolin 1α in mouse lung and kidney at various developmental time points (30× cycles). Lane w: water; 11: E11.5; 15: E15.5; 18: E18.5; nb: newborn; ad: adult. B: QRT‐PCR for ACE2 expression in mouse lung and kidney at various developmental time points, normalized to the 18S amplicon. nb: newborn. *P = 0.001 when compared to E17.5.