Calpain3 is expressed in a proteolitically active form in papillomavirus-associated urothelial tumors of the urinary bladder in cattle

Abstract

Background: Calpain 3 (Capn3), also named p94, is a skeletal muscle tissue-specific protein known to be responsible for limb-girdle muscular dystrophy type 2A (LGMD2A). Recent experimental studies have hypothesized a pro-apoptotic role of Capn3 in some melanoma cell lines. So far the link between calpain3 and tumors comes from in vitro studies. The objective of this study was to describe Capn3 activation in naturally occurring urothelial tumors of the urinary bladder in cattle.

Methods and findings: Here we describe, for the first time in veterinary and comparative oncology, the activation of Capn3 in twelve urothelial tumor cells of the urinary bladder of cattle. Capn3 protein was initially identified with nanoscale liquid chromatography coupled with tandem mass spectrometry (nano LC-MS/MS) in a co-immunoprecipitation experiment on E2F3, known to be a transcription factor playing a crucial role in bladder carcinogenesis in humans. Capn3 expression was then confirmed by reverse transcription polymerase chain reaction (RT-PCR). Finally, the Ca(2+)-dependent proteolytic activity of Capn3 was assayed following ion exchange chromatography. Morphologically, Capn3 expression was documented by immunohistochemical methods. In fact numerous tumor cells showed an intracytoplasmic immunoreactivity, which was more rarely evident also at nuclear level. In urothelial tumors, bovine papillomavirus type 2 (BPV-2) DNA was amplified by PCR and the expression of E5 protein, the major oncogenic protein of BVP-2, was detected by western blotting, immunohistochemistry, and immunofluorescence. E2F3 overexpression and pRb protein downregulation were shown by western blotting.

Conclusion: The role of capn3 protein in urothelial cancer of the urinary bladder remains to be elucidated: further studies would be required to determine the precise function of this protease in tumor development and progression. However, we suggest that activated Capn3 may be involved in molecular pathways leading to the overexpression of E2F3, which in turn could be responsible for urothelial tumor cell proliferation also in cattle, though other mechanisms are likely to exist. If further studies corroborate the important role of Capn3 in urothelial tumors of the urinary bladder, cattle with urinary tumors may prove useful as animal model for bladder carcinogenesis.

Figure 1. Expression of activated Capn3 in urothelial tumors.

Detection of Capn3 transcript in bovine bladders. PCR analysis was performed on the cDNAs synthesized from bladders isolated from different animals as reported in Methods. The primer pair utilized to detect Capn3 was Sn831/Asn1845 and the conditions are reported in Methods. PCR products were separated on 1.2% agarose gel. Each lane corresponds to a different animal. The lanes having the same letter refer to the same animal. cDNA amounts are normalized with respect to equal GAPDH levels.

Figure 2. Expression of activated Capn3 in urothelial tumors.

Detection of IS1 and IS2 inserts in Capn3 transcript. PCR analysis was performed on the cDNAs synthesized from bladders isolated from pathological animals or human PBMC as reported in Methods. The primer pair utilized to detect Capn3 IS1 insert was Sn831/Asn1845 (A) and that utilized to detect IS2 insert was Sn1884/Asn2669 (B). PCR conditions are reported in Methods. PCR products were separated by electrophoresis on 1.2% agarose gel. Marker sizes: GeneRuler 100 bp DNA ladder (Fermentas). cDNA amounts are normalized with respect to equal GAPDH levels. The expected sizes for the PCR fragments in (A) are 1018 bp with IS1, and 829 bp without IS1. The expected sizes for the PCR fragments in (B) are 805 bp with IS2, and 577 bp without IS2.

Figure 3. Expression of activated Capn3 in urothelial tumors.

Ion-exchange chromatography on bovine bladders. Crude extracts were prepared from normal (full circles) or pathological (empty circles) bovine bladders as reported in methods. Aliquots (35 mg) were submitted to ion-exchange chromatography and calpain activity was assayed on aliquots (100 µl) of the eluted fractions as described in Methods.

Figure 4. Expression of activated Capn3 in urothelial tumors.

Immunohistochemical detection of Capn3. Capn3 is not expressed in normal urothelium. Streptavidin-biotin-peroxidase, Mayer's hematoxylin counterstain.

Figure 5. Expression of activated Capn3 in urothelial tumors.

A weak immunoreactivity can be seen in urothelial cells of the grossly unaffected mucosa. Streptavidin-biotin-peroxidase, Mayer's hematoxylin counterstain.

Figure 6. Expression of activated Capn3 in urothelial tumors.

Capn3 expression is mostly evident in the cytoplasm of basal neoplastic urothelial cells. Streptavidin-biotin-peroxidase, Mayer's hematoxylin counterstain.

Figure 7. Expression of activated Capn3 in urothelial tumors.

Capn3 expression is also detected in some nuclei. Streptavidin-biotin-peroxidase, Mayer's hematoxylin counterstain.

Figure 8. Expression of activated Capn3 in urothelial tumors.

PCR amplification of urinary bladder samples. Lanes:1–3, tumor samples; 4, positive control of BPV-2 plasmid; 5–6, negative control with no DNA added; M, molecular mass marker #8 (Roche, Milan, Italy).

Figure 9. Expression of activated Capn3 in urothelial tumors.

E5 oncoprotein expression detected by immunoprecipitation. Lane 1, negative normal bladder tissue from healthy cattle; lanes 2–6 neoplastic samples showing an evident E5 expression except in lane 2.

Figure 10. Expression of activated Capn3 in urothelial tumors.

E5 oncoprotein documented by scanning laser confocal microscope. Immunofluorescence is evident in the cytoplasm of several neoplastic cells (arrows).

Figure 11. Expression of activated Capn3 in urothelial tumors.

Western blot analysis to characterize the expression of E2F3. Lane 1, healthy animal as control; lanes 2–7, E2F3 protein appears to be overexpressed in all examined tumor samples.

Figure 12. Expression of activated Capn3 in urothelial tumors.

Western blot analysis to characterize the expression of pRb and constitutive SerpinA3-7 protein. A downregulation of pRb protein expression is severely evident in all tumor samples. The SerpinA3-7 protein was normally expressed in tumor samples.