Expression and activation of caspase-6 in human fetal and adult tissues

Abstract

Caspase-6 is an effector caspase that has not been investigated thoroughly despite the fact that Caspase-6 is strongly activated in Alzheimer disease brains. To understand the full physiological impact of Caspase-6 in humans, we investigated Caspase-6 expression. We performed western blot analyses to detect the pro-Caspase-6 and its active p20 subunit in fetal and adult lung, kidney, brain, spleen, muscle, stomach, colon, heart, liver, skin, and adrenals tissues. The levels were semi-quantitated by densitometry. The results show a ubiquitous expression of Caspase-6 in most fetal tissues with the lowest levels in the brain and the highest levels in the gastrointestinal system. Caspase-6 active p20 subunits were only detected in fetal stomach. Immunohistochemical analysis of a human fetal embryo showed active Caspase-6 positive apoptotic cells in the dorsal root ganglion, liver, lung, kidney, ovary, skeletal muscle and the intestine. In the adult tissues, the levels of Caspase-6 were lower than in fetal tissues but remained high in the colon, stomach, lung, kidney and liver. Immunohistological analyses revealed that active Caspase-6 was abundant in goblet cells and epithelial cells sloughing off the intestinal lining of the adult colon. These results suggest that Caspase-6 is likely important in most tissues during early development but is less involved in adult tissues. The low levels of Caspase-6 in fetal and adult brain indicate that increased expression as observed in Alzheimer Disease is a pathological condition. Lastly, the high levels of Caspase-6 in the gastrointestinal system indicate a potential specific function of Caspase-6 in these tissues.

Figure 1. Steady state levels of Casp6, Casp1, and Casp3 in human fetal tissues.

A. Representative western blot (series #1) of 100 µg of total proteins from fetal tissues with Upstate anti-Casp6 antisera (ProCasp6), neoepitope 10630 antisera (p20Casp6), and β-Actin antibody. B. Fetal age for the three series of tissues used in the study. C. Bar graph of proCasp6 levels relative to that in colon arbitrarily placed at 100. Data represent the individual values from series 1,2, and 3, as well as mean and SEM of 3 independent experiments. D. Coomassie stained gel of proteins from tissues in series 1. E –G. Representative western blots of 100 µg of total proteins from fetal tissues with E. anti-Casp1 antisera (ProCasp1), neoepitope antisera to the p20 subunit of Casp1 (p20Casp1) and β-Actin antibody, F. anti-Casp3 antisera (ProCasp3), neoepitope antisera to the p20 subunit of Casp3 (p20Casp3) and β-Actin antibody, and G. Western blot of 100 µg of total proteins from three different fetal brains, primary cultures of human fetal neurons or astrocytes with Upstate anti-Casp6 antisera (ProCasp6) or β-actin antibody.

Figure 2. Steady state levels of Casp6, Casp1, and Casp3 in 8 different human fetal stomachs.

Representative western blots containing 100 µg total protein/lane from fetal stomachs of different developmental ages with A. Neomarker anti-Casp6 antisera (top panel), neoepitope 10630 antisera (second panel), and β-Actin antibody (third panel), B. Micrograph of human fetal stomach stained with 10630 anti-active Casp6 antiserum. C& D. Western blot of (C) anti-Casp1 antisera (ProCasp1), neoepitope antisera to the p20 subunit of Casp1 (p20Casp1) and β-Actin antibody, and (D) anti-Casp3 antisera (ProCasp3), neoepitope antisera to the p20 subunit of Casp3 (p20Casp3) and β-Actin antibody.

Figure 3. Micrographs of immunohistochemical analyses of fetal tissues with 1277 neoepitope antisera detecting the active p20 subunit of Casp6.

A. At low power (original magnification, 100x), immunopositive cells (arrows) in the dorsal root ganglion (DRG), in the anterior horn (AH), and in the dorsal root entry zone (DREZ) of the spinal cord white matter (WM). B. High power view of dorsal root ganglion (DRG: original magnification, 400×) indicating mitotic activity (solid arrowhead), features of early neuronal differentiation (open arrowheads) and several apoptotic immunopositive cells (arrows). C. High power view of anterior horn (AH: original magnification, 400×) indicating dividing cells (solid arrow heads), early maturation of cells (open arrowheads), and an immunopositive apoptotic precursor cell (arrow). WM; white matter). D. High power view of dorsal spinal cord (original magnification, 400×). Immunopositive finely granular (synaptic) pattern in the dorsal root entry zone (DREZ) of the spinal cord white matter (WM). DRG, dorsal root ganglion; DR, dorsal root; PH, posterior horn). E. Extensive extramedullary hematopoiesis (EMH) in the sinusoids that separate the cords of hepatocytes (HC) in the liver (original magnification 400×). In the hematopoietic islands, there are many immunopositive apoptotic cells (arrows).

Figure 4. Steady state levels of Casp6, Casp1, and Casp3 in human adult tissues.

A. Representative western blot (series #2) of 100 µg of total proteins from adult tissues with Upstate anti-Casp6 antisera (ProCasp6) and β-Actin antibody. B. Age of adult tissues used for the study. C. Bar graph of the average of proCasp6 levels relative to that in colon arbitrarily placed at 100. Data represent the individual values as well as mean and SEM of 3 independent experiments. D. Coomassie stained gel of proteins from tissues in series 2.

Figure 5. Micrographs of immunohistochemical analyses of adult normal and cancerous colon with 1277 neoepitope antisera against the active p20 subunit of Casp6.

Normal colonic mucosa (A) and crypt openings (B), colonic adenocarcinomas (C) and crypt openings (D) immunostained with anti-p20Casp6 neoepitope antisera. E. Larger magnification of normal colon showing active Casp6-positive epithelial cell sloughing off in the lumen. F. Western blot of 100 µg of total proteins from normal and cancerous colon tissues with Upstate anti-Casp6 antisera (ProCasp6) and β-Actin antibody.