Disruption of the murine calpain small subunit gene, Capn4: calpain is essential for embryonic development but not for cell growth and division

Abstract

Calpains are a family of Ca(2+)-dependent intracellular cysteine proteases, including the ubiquitously expressed micro- and m-calpains. Both mu- and m-calpains are heterodimers, consisting of a distinct large 80-kDa catalytic subunit, encoded by the genes Capn1 and Capn2, and a common small 28-kDa regulatory subunit (Capn4). The physiological roles and possible functional distinctions of mu- and m-calpains remain unclear, but suggested functions include participation in cell division and migration, integrin-mediated signal transduction, apoptosis, and regulation of cellular control proteins such as cyclin D1 and p53. Homozygous disruption of murine Capn4 eliminated both mu- and m-calpain activities, but this did not affect survival and proliferation of cultured embryonic stem cells or embryonic fibroblasts, or the early stages of organogenesis. However, mutant embryos died at midgestation and displayed defects in the cardiovascular system, hemorrhaging, and accumulation of erythroid progenitors.

FIG. 1

Targeted disruption of the calpain small subunit gene. Schematic representations show the murine Capn4 allele (upper), the targeting vector (middle), and the targeted allele (lower). The Capn4 gene was cloned from the 129SvJ mouse strain (1). Positions of PstI (P), PvuII (Pv), BamHI (B), and HindIII (H) restriction endonuclease sites are indicated. Homologous recombination replaces a 441-bp PvuII-BamHI fragment containing the 3′ end of Capn4 exon 9 with the neo cassette. Tk, thymidine kinase.

FIG. 2

Analysis of ES cells. (A) Southern blotting. A 487-bp exon 5/6-containing PvuII fragment (Fig. 1) was used as a probe in Southern blot hybridization analysis of DNA isolated from Capn4^+/+, Capn4^+/−, and Capn4^−/− ES cells. PstI fragments of 5.5 or 2.5 kb are diagnostic of the wild-type or targeted Capn4 allele, respectively. (B) Northern blotting. Equal amounts of poly(A)⁺ RNA were electrophoresed on 1.2% formaldehyde agarose gels and transferred to nylon membranes. The membranes were probed with [α-³²P]dATP-labeled cDNA fragments specific for the mouse μ- and m-calpain large subunits and the mouse calpain small subunit. The final washes were in 0.1× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate)–0.1% SDS at 52°C. Equal gel loading was confirmed both by ethidium bromide staining and by stripping the blots and reprobing with an actin cDNA. (C) Casein zymogram. ES cells were lysed in lysis buffer (see Materials and Methods). The lysates were centrifuged to remove cell debris, and portions of the supernatants containing 30 μg of protein were run on casein zymograms. After electrophoresis, the gels were rinsed and incubated overnight in the presence of 5 mM Ca²⁺ and 10 mM 2-mercaptoethanol. (D and E) Immunoblot analysis. Equal amounts of protein denatured in SDS sample buffer were run on SDS-polyacrylamide gels (9%) in Tris-Tricine buffer and then blotted onto Immobilon. The calpain m-calpain large subunit (80 kDa) (D) and the control Erk1/2 kinase proteins (42 and 44 kDa) (E) were detected with specific antibodies, followed by horseradish peroxidase-labeled second antibodies and enhanced chemiluminescence detection. The Capn4 genotype of the cells represented in each lane is shown as +/+, +/−, or −/−.

FIG. 3

ES cell growth curves. Several six-well plates were prepared with ES cells grown in ES medium. The cells were harvested with trypsin at 20- to 24-h intervals up to 96 h and counted by means of a Coulter counter. The data points and error bars (not all of which are visible) indicate the average cell number and range of three observations. The lines are exponential curves fitted to the data for Capn4^+/+ (●), Capn4^+/− (▿), and Capn4^−/− (■) ES cells.

FIG. 4

Analysis of E10.5 embryos. (A) Genotypes of individual embryos were determined by Southern blotting analysis of DNA from yolk sacs as described in the legend to Fig. 2A. No result was obtained for embryo 3, which is, however, seen in panels B and C to be either +/+ or +/−. (B) Calpain activity determined in individual embryo lysates as described in the legend to Fig. 2C. (C and D) Immunoblotting analysis of mouse m-calpain large subunit (C) and Erk1/2 kinase (D), performed as described in the legend to Fig. 2.

FIG. 5

Histological analysis of E10.5 and E11.5 embryos. (A to D) Midsagittal sections showing the heart, branchial arches, dorsal aorta, sinus venosus, and liver primordium. (E to H) Sagittal sections through the heart showing ventricle (left) and atrium (right). (I to L) Higher-power magnification of heart sagittal sections showing the atrial wall (upper) and a portion of the ventricle (lower). (M to P) Sagittal sections through the liver primordium. (Q to T) Sagittal sections through the fourth ventricle (right) and adjacent trigeminal neural crest (left). (A, E, I, M, and Q) Capn4^+/+ E10.5 embryos; (B, F, J, N, and R) Capn4^−/− E10.5 embryos; (C, G, K, O, and S) Capn4^+/+ E11.5 embryos; (D, H, L, P, and T) Capn4^−/− E11.5 embryos.