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

• Published in: Peptides (New York, N.Y. : 1980) N.Y. : 1980), 2009-10, Vol.30 (10), p.1882-1891
• Main Authors: Beinfeld, Margery C., Funkelstein, Lydiane, Foulon, Thierry, Cadel, Sandrine, Kitagawa, Kouki, Toneff, Thomas, Reinheckel, Thomas, Peters, Christoph, Hook, Vivian
• Format: Article
• Language: English
• Subjects: Adrenocorticotropic Hormone - metabolism; Aminopeptidases - antagonists & inhibitors; Aminopeptidases - genetics; Aminopeptidases - metabolism; Animals; Biological and medical sciences; Brain; Cathepsin L; Cathepsin L - antagonists & inhibitors; Cathepsin L - genetics; Cathepsin L - metabolism; Cells, Cultured - metabolism; Cerebral Cortex - cytology; Cerebral Cortex - metabolism; Cholecystokinin; Cholecystokinin - metabolism; Fundamental and applied biological sciences. Psychology; Lysosomal-Associated Membrane Protein 1 - metabolism; Mice; Mice, Knockout; Pituitary; Pituitary Gland - cytology; Pituitary Gland - metabolism; Prohormone convertase; Proprotein Convertases - antagonists & inhibitors; Proprotein Convertases - genetics; Proprotein Convertases - metabolism; Protease; Protein Isoforms - genetics; Protein Isoforms - metabolism; Protein Precursors - genetics; Protein Precursors - metabolism; RNA, Small Interfering - genetics; RNA, Small Interfering - metabolism; Vertebrates: endocrinology
• ISSN: 0196-9781; 1873-5169
• DOI: 10.1016/j.peptides.2009.06.030

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 NH 2-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.