Cathepsin L plays a major role in cholecystokinin production in mouse brain cortex and in pituitary AtT-20 cells: protease gene knockout and inhibitor studies.
Cholecystokinin (CCK) is a peptide neurotransmitter whose production requires proteolytic processing of the proCCK precursor to generate active CCK8 neuropeptide in brain. This study demonstrates the significant role of the cysteine protease cathepsin L for CCK8 production. In cathepsin L knockout (KO) mice, CCK8 levels were substantially reduced in brain cortex by an average of 75%. To evaluate the role of cathepsin L in producing CCK in the regulated secretory pathway of neuroendocrine cells, pituitary AtT-20 cells that stably produce CCK were treated with the specific cathepsin L inhibitor, CLIK-148. CLIK-148 inhibitor treatment resulted in decreased amounts of CCK secreted from the regulated secretory pathway of AtT-20 cells. CLIK-148 also reduced cellular levels of CCK9 (Arg-CCK8), consistent with CCK9 as an intermediate product of cathepsin L, shown by the decreased ratio of CCK9/CCK8. The decreased CCK9/CCK8 ratio also suggests a shift in the production to CCK8 over CCK9 during inhibition of cathepsin L. During reduction of the PC1/3 processing enzyme by siRNA, the ratio of CCK9/CCK8 was increased, suggesting a shift to the cathepsin L pathway for the production of CCK9. The changes in ratios of CCK9 compared to CCK8 are consistent with dual roles of the cathepsin L protease pathway that includes aminopeptidase B to remove NH2-terminal Arg or Lys, and the PC1/3 protease pathway. These results suggest that cathepsin L functions as a major protease responsible for CCK8 production in mouse brain cortex, and participates with PC1/3 for CCK8 production in pituitary cells.
Beinfeld
Margery C
MC
Department of Pharmacology and Experimental Therapeutics, Tufts University School of Medicine, 136 Harrison Ave, Boston, MA 02111, United States.
Funkelstein
Lydiane
L
Foulon
Thierry
T
Cadel
Sandrine
S
Kitagawa
Kouki
K
Toneff
Thomas
T
Reinheckel
Thomas
T
Peters
Christoph
C
Hook
Vivian
V
D000324:Adrenocorticotropic Hormone / Q000378:metabolism; D000626:Aminopeptidases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D000818:Animals; D056668:Cathepsin L / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured / Q000378:metabolism; D002540:Cerebral Cortex / Q000166:cytology / Q000378:metabolism*; D002766:Cholecystokinin / Q000378:metabolism*; D052118:Lysosomal-Associated Membrane Protein 1 / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D010902:Pituitary Gland / Q000166:cytology* / Q000378:metabolism; D043484:Proprotein Convertases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism*; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism
D052118:Lysosomal-Associated Membrane Protein 1; D020033:Protein Isoforms; D011498:Protein Precursors; D034741:RNA, Small Interfering; D000324:Adrenocorticotropic Hormone; D002766:Cholecystokinin; D000626:Aminopeptidases; C019721:aminopeptidase B; D043484:Proprotein Convertases; D056668:Cathepsin L