Monitoring of epithelial cell caspase activation via detection of durable keratin fragment formation.

Keratins 18 and 19 (K18/K19) are epithelial-specific intermediate filament proteins. Apoptosis induces caspase cleavage at the highly conserved K18 or K19 Asp237, which in K18 is preceded by cleavage at Asp396. We characterized the keratin N-terminal fragments that are generated upon caspase digestion of K18/K19 at Asp237 in order to study keratin dynamics during apoptosis. This was carried out by generating and characterizing antibodies selective to K18/K19 Asp237. K18 or K19 peptides that expose Asp237 in 234VEVD were used for rabbit immunization. The generated antibodies recognized cleaved but not intact K18/K19, exclusively, as determined by blotting or immunofluorescence staining of apoptotic human HT29 cells or livers isolated from Fas-Ab-injected mice. Antibodies to K18/K19 Asp237 recognized the common VEVD-motif as determined by immunoblotting of cells transfected with K18, K19 or K20. The K18/K19 VEVD-directed antibodies demonstrated sequential Asp396 then Asp237 K18 cleavage during apoptosis. Specific-keratin selectivity of the anti-Asp237 antibodies was confirmed by their inability to recognize K14 after UV-induced apoptosis in transfected cells. The Asp237-containing apoptotic keratin fragments are secreted into the medium of cultured HT29 cells and are stable up to 96 h after inducing apoptosis. Furthermore, the generated antibodies recognize keratin apoptotic fragments in sera of mice undergoing hepatocyte apoptosis and sera of patients with cirrhosis, and also recognize apoptotic cells in various epithelial human tumours. Therefore, the N-terminal caspase-generated K18 fragment is stable in tissues and biological fluids. The Asp237-directed antibodies provide a powerful tool to study apoptosis in human and mouse tissues, cells and serum, using a broad range of detection modalities.

A peptide derived from pICln induced a strong hypotonic resistance in Escherichia coli cells.

We demonstrated previously that expression of rat pICln in Escherichia coli conferred a strong resistance to hypotonic stress. To define the intramolecular functional domain responsible for the resistance, molecular dissection of pICln was performed and the obtained peptides were expressed in E. coli. The cells expressing the peptides were exposed to a hypotonic solution, and their 'survival rates' were observed. The cells expressing only the peptides including the second acidic domain of pICln exhibited significantly higher 'survival rates' after hypotonic stress. The functional domain against hypotonicity was finally narrowed down to a peptide consisting of a 46-amino acid residue, P107-152. We conclude that the expression of P107-152 in E. coli cells could enhance their resistance to a hypotonic environment.

Expression of pI(Cln) in Escherichia coli gives a strong tolerance to hypotonic stress.

We amplified the coding region DNA sequence from a rat renal pI(Cln) cDNA by PCR and expressed the protein in Escherichia coli cells. The cells were exposed to hypotonic conditions followed by spreading them onto LB plates for subsequent colony survival assay. The present study demonstrated that the cells expressing pI(Cln) exhibit a strong resistance to hypotonic stress. Moreover, the resistance was specifically inhibited by extracellular ATP and some anion channel inhibitors. These findings indicate that the expression of pI(Cln) directly confers tolerance to hypotonic stress, and pI(Cln) is concluded to be an important molecule for cell-volume regulation.

pICln predominantly localizes at luminal surface membranes of distal tubules and Henle's ascending limbs.

We produced a highly specific antibody to the C-terminal peptide sequence of pICln. It recognized pICln with a 38-kDa molecular mass on SDS-polyacrylamide gel electrophoresis, coinciding with that previously reported. During native polyacrylamide gel electrophoresis, three immunoreactive bands (38, 70, and 130 kDa) were detected. The isoelectric point of pICln was calculated to be 4.0. Subcellular localization study showed the presence of pICln in the soluble and microsomal fraction. pICln can be easily solubilized from the membrane fraction with Triton X-100. From immunohistochemical observations, we found pICln to be obviously located on the luminal surface membranes of the distal tubules and Henle's loop ascending limbs, and it can also be found inside proximal tubular cells. The present results suggested that pICln functions as a "cytosolic anchor = membrane insertion" model, and it plays important roles in the "urine dilution segment" cells of nephrons.