The neutral cysteine protease bleomycin hydrolase is essential for epidermal integrity and bleomycin resistance

Abstract

The papain superfamily member bleomycin hydrolase (Blmh) is a neutral cysteine protease with structural similarity to a 20S proteasome. Bleomycin (BLM), a clinically used glycopeptide anticancer agent, is deaminated in vitro by Blmh. We used gene targeting to generate mice that lack Blmh and demonstrated that Blmh is the sole enzyme required for BLM deamination. Although some Blmh null mice were viable and reproduced, only about 65% of the expected number survived the neonatal period, revealing an important role for Blmh in neonatal survival. Mice lacking Blmh exhibited variably penetrant tail dermatitis that resembled rodent ringtail. The histopathology of the tail dermatitis was similar to skin lesions in humans with pellagra, necrolytic migratory erythema, and acrodermatitis enteropathica. Compared with controls, Blmh null mice were more sensitive to acute BLM lethality and developed pulmonary fibrosis more readily following BLM treatment. Thus, we have established that Blmh is an essential protectant against BLM-induced death and has an important role in neonatal survival and in maintaining epidermal integrity.

Figure 1

Targeted disruption of the Blmh gene. (A) A replacement vector (not shown) was constructed with two fragments (hatched) of wild-type Blmh genomic DNA (a) flanking the neo gene. ∗, Exon 3 encodes (in part) GRCWIF, which is the conserved amino acid sequence surrounding the catalytic cysteine (italicized). (b) A targeted Blmh allele. PCR primers, p1, p2, and p3; B, BglII; BH1, BamHI; H, HindIII; P, PstI; RI, EcoRI; X, XbaI. (B) Identification of the targeted Blmh locus using Southern blot (a) and three-primer PCR (b) analyses of mouse tail (M) or ES cell DNA. Genomic DNA from heterozygous (+/−) ES cells that contained either Blmh^tm1Geh or Blmh^tm2Geh targeted alleles, wild-type parental R1 ES cells (R1), and Bh−/− (−/−), or Bh+/+ (+/+) mice were digested with BglII and hybridized with probe A (a). (C) Blmh mRNA expression in embryos using Northern blot analysis. Total RNA from Bh+/+ (+/+), Bh+/− (+/−), and Bh−/− (−/−) embryos was hybridized to probe A (see A, b) or β-actin cDNA. (D) Blmh enzymatic activity in lung homogenates from Bh+/+ and Bh−/− mice was determined by resolving BLM A₂ (A₂) from its metabolite desamido-BLM A₂ (dA₂) by reverse-phase HPLC. Representative HPLC tracings are shown from a reaction containing BLM A₂ (a), Bh+/+ lung homogenates supplemented with either diluent (b), substrate (c), or substrate and the cysteine protease inhibitor E-64 (d), and Bh−/− lung homogenate plus substrate (e). Note the lack of desamido-BLM A₂ or an alternative metabolite after BLM A₂ was exposed to Bh−/− homogenate (e).

Figure 2

Gross appearance and histopathology of tail dermatitis on Blmh null mice. (A) The gross appearance of 18-day-old wild-type (+/+) and Blmh null (−/−) littermates. Dermatitis is apparent only on the tail of Blmh null pups. (B) Tails from adult wild-type (+/+) and Blmh null (−/−) mice. (C–F) Hematoxylin- and eosin-stained cross sections (6 μm) of tail biopsies from Bh+/+ (C) and Bh−/− (D–F) mice. The arrowheads in D and E identify ballooning degeneration (BD) of the stratum spinosum. (F) Full thickness necrosis and abundant neutrophil infiltrate (arrow), from a Blmh null mouse afflicted with severe dermatitis. SB, stratum basale; SC, stratum corneum; SG, stratum granulosum; SS, stratum spinosum; PK, parakeratosis. (Bars = 25 μm.)

Figure 3

Low-humidity-induced dermatitis in Bh−/− mice following 20 days in humidity-controlled chambers. Gross appearance of Blmh null (−/−) and wild-type (+/+) littermates following exposure to either high (A; 80.1 ± 1.2%) or low (B; 20.7 ± 0.6%) relative humidity.

Figure 4

Hydroxyproline (HO-pro) content of right lung 6 weeks after a 7-day continuous s.c. infusion of BLM or saline. Bh+/+ and Bh−/− mice received saline (n = 4) or BLM (10 mg/kg, n = 4; or 25 mg/kg, n = 5). Results are shown as mean ± SEM. ∗, P < 0.05, Student’s t test: BLM vs. saline groups of Bh−/− mice.