{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,22]],"date-time":"2026-10-22T20:19:40Z","timestamp":1792700380207,"version":"4.5.0"},"reference-count":32,"publisher":"MDPI AG","issue":"11","license":[{"start":{"date-parts":[[2023,5,26]],"date-time":"2023-05-26T00:00:00Z","timestamp":1685059200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001659","name":"DFG","doi-asserted-by":"publisher","award":["BR 1308\/11-2"],"award-info":[{"award-number":["BR 1308\/11-2"]}],"id":[{"id":"10.13039\/501100001659","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>In the thyroid gland, cysteine cathepsins are secreted upon thyrotropin stimulation for thyroglobulin processing, and they are present at the primary cilia of thyroid epithelial cells. Treatment with protease inhibitors resulted in the loss of cilia from rodent thyrocytes and caused redistribution of the thyroid co-regulating G protein-coupled receptor Taar1 to the endoplasmic reticulum. These findings suggest that ciliary cysteine cathepsins are important to maintain sensory and signaling properties for the proper regulation and homeostasis of thyroid follicles. Therefore, it is important to better understand how cilia structure and frequencies are maintained in human thyroid epithelial cells. Hence, we aimed to investigate the potential role of cysteine cathepsins for the maintenance of primary cilia in the normal human Nthy-ori 3-1 thyroid cell line. This was approached by determining cilia lengths and frequencies in cysteine peptidase inhibition conditions in Nthy-ori 3-1 cell cultures. Cilia lengths were shortened upon 5 h of cysteine peptidase inhibition with cell-impermeable E64. Likewise, cilia lengths and frequencies were decreased upon additional overnight treatment with the cysteine peptidase-targeting, activity-based probe DCG-04. The results suggest that cysteine cathepsin activity is required for the maintenance of the cellular protrusions not only in rodents, but also in human thyrocytes. Hence, thyrotropin stimulation was used to simulate physiological conditions that eventually lead to cathepsin-mediated thyroglobulin proteolysis, which is initiated in the thyroid follicle lumen. Immunoblotting revealed that thyrotropin stimulation conditions result in the secretion of little procathepsin L and some pro- and mature cathepsin S but no cathepsin B from the human Nthy-ori 3-1 cells. Unexpectedly, however, 24 h incubation periods with thyrotropin shortened the cilia although higher amounts of cysteine cathepsins were present in the conditioned media. These data point to the necessity of further studies to delineate which of the cysteine cathepsins plays the most prominent role in cilia shortening and\/or elongation. Collectively, the results of our study provide corroboration for the hypothesis of thyroid autoregulation by local mechanisms that our group previously proposed.<\/jats:p>","DOI":"10.3390\/ijms24119292","type":"journal-article","created":{"date-parts":[[2023,5,26]],"date-time":"2023-05-26T02:05:44Z","timestamp":1685066744000},"page":"9292","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":6,"title":["Investigations on Primary Cilia of Nthy-ori 3-1 Cells upon Cysteine Cathepsin Inhibition or Thyrotropin Stimulation"],"prefix":"10.3390","volume":"24","author":[{"ORCID":"https:\/\/orcid.org\/0009-0007-7215-5035","authenticated-orcid":false,"given":"Alara Gaye","family":"Do\u011fru","sequence":"first","affiliation":[{"name":"School of Science, Constructor University, Campus Ring 1, D-28759 Bremen, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7854-0977","authenticated-orcid":false,"given":"Maren","family":"Rehders","sequence":"additional","affiliation":[{"name":"School of Science, Constructor University, Campus Ring 1, D-28759 Bremen, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4476-0357","authenticated-orcid":false,"given":"Klaudia","family":"Brix","sequence":"additional","affiliation":[{"name":"School of Science, Constructor University, Campus Ring 1, D-28759 Bremen, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2023,5,26]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"923","DOI":"10.1515\/bc.2010.109","article-title":"Nuclear cysteine cathepsin variants in thyroid carcinoma cells","volume":"391","author":"Tedelind","year":"2010","journal-title":"Biol. Chem."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"194","DOI":"10.1016\/j.biochi.2007.07.024","article-title":"Cysteine cathepsins: Cellular roadmap to different functions","volume":"90","author":"Brix","year":"2008","journal-title":"Biochimie"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"437","DOI":"10.1055\/a-1080-2969","article-title":"Auto-Regulation of the Thyroid Gland Beyond Classical Pathways","volume":"128","author":"Brix","year":"2020","journal-title":"Exp. Clin. Endocrinol. Diabetes"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"355","DOI":"10.1152\/physrev.00030.2013","article-title":"Thyroid Hormone Regulation of Metabolism","volume":"94","author":"Mullur","year":"2014","journal-title":"Physiol. Rev."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"1733","DOI":"10.1172\/JCI15990","article-title":"Thyroid functions of mouse cathepsins B, K, and L","volume":"111","author":"Friedrichs","year":"2003","journal-title":"J. Clin. Investig."},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Jordans, S., Jenko-Kokalj, S., K\u00fchl, N., Tedelind, S., Sendt, W., Br\u00f6mme, D., Turk, D., and Brix, K. (2009). Monitoring compartment-specific substrate cleavage by cathepsins B, K, L, and S at physiological pH and redox conditions. BMC Biochem., 10.","DOI":"10.1186\/1471-2091-10-23"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"118846","DOI":"10.1016\/j.bbamcr.2020.118846","article-title":"Significance of nuclear cathepsin V in normal thyroid epithelial and carcinoma cells","volume":"1867","author":"Venugopalan","year":"2020","journal-title":"Biochim. Biophys. Acta Mol. Cell Res."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"211","DOI":"10.1007\/s12079-017-0436-0","article-title":"The primary cilium\u2014Once a \u201crudimentary\u201d organelle that is now a ubiquitous sensory cellular structure involved in many pathological disorders","volume":"12","author":"Wheatley","year":"2017","journal-title":"J. Cell Commun. Signal."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"595","DOI":"10.1089\/thy.2018.0401","article-title":"Primary Cilium in the Human Thyrocyte: Changes in Frequency and Length in Relation to the Functional Pathology of the Thyroid Gland","volume":"29","author":"Utrilla","year":"2019","journal-title":"Thyroid"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"270","DOI":"10.1016\/j.biochi.2019.07.010","article-title":"Treatment of rat thyrocytes in vitro with cathepsin B and L inhibitors results in disruption of primary cilia leading to redistribution of the trace amine associated receptor 1 to the endoplasmic reticulum","volume":"166","author":"Szumska","year":"2019","journal-title":"Biochimie"},{"key":"ref_11","unstructured":"Brix, K., Qatato, M., Szumska, J., Venugopalan, V., and Rehders, M. (2019). The Thyroid and Its Diseases, Springer Nature Switzerland AG."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"8","DOI":"10.3389\/fcell.2018.00008","article-title":"Signaling through the Primary Cilium","volume":"6","author":"Wheway","year":"2018","journal-title":"Front. Cell Dev. Biol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"12871","DOI":"10.1073\/pnas.1323292111","article-title":"Primary cilia signaling mediates intraocular pressure sensation","volume":"111","author":"Luo","year":"2014","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"700083","DOI":"10.3389\/fendo.2021.700083","article-title":"Primary Cilia Mediate TSH-Regulated Thyroglobulin Endocytic Pathways","volume":"12","author":"Lee","year":"2021","journal-title":"Front. Endocrinol."},{"key":"ref_15","first-page":"924","article-title":"TSH Signal Transduction in Thyroid Cells of Nthy-Ori 3\u20131 Line","volume":"11","author":"Piotrowska","year":"2011","journal-title":"Post\u0119py Nauk. Med."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"569","DOI":"10.1016\/S1074-5521(00)00014-4","article-title":"Epoxide electrophiles as activity-dependent cysteine protease profiling and discovery tools","volume":"7","author":"Greenbaum","year":"2000","journal-title":"Chem. Biol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"R100","DOI":"10.1186\/gb-2006-7-10-r100","article-title":"CellProfiler: Image analysis software for identifying and quantifying cell phenotypes","volume":"7","author":"Carpenter","year":"2006","journal-title":"Genome Biol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"773","DOI":"10.1515\/BC.2002.081","article-title":"Thyroid Stimulating Hormone Upregulates Secretion of Cathepsin B from Thyroid Epithelial Cells","volume":"383","author":"Linke","year":"2002","journal-title":"Biol. Chem."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"238","DOI":"10.1177\/154411130201300304","article-title":"Cysteine Peptidases of Mammals: Their Biological Roles and Potential Effects in the Oral Cavity and Other Tissues in Health and Disease","volume":"13","author":"Dickinson","year":"2002","journal-title":"Crit. Rev. Oral Biol. Med."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"31736","DOI":"10.1074\/jbc.M114.609396","article-title":"A Role for Stefin B (Cystatin B) in Inflammation and Endotoxemia","volume":"289","author":"Maher","year":"2014","journal-title":"Biol. Chem."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"33194","DOI":"10.1074\/jbc.M302742200","article-title":"Biosynthetic processing of cathepsins and lysosomal degradation are abolished in asparaginyl endopeptidase-deficient mice","volume":"278","author":"Yamamoto","year":"2003","journal-title":"Biol. Chem."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"126","DOI":"10.1016\/j.biochi.2015.09.022","article-title":"Structure and function of legumain in health and disease","volume":"122","author":"Dall","year":"2016","journal-title":"Biochimie"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"4487","DOI":"10.1242\/jcs.113.24.4487","article-title":"Cathepsin K in thyroid epithelial cells: Sequence, localization and possible function in extracellular proteolysis of thyroglobulin","volume":"113","author":"Tepel","year":"2000","journal-title":"J. Cell Sci."},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Dauth, S., S\u00eerbulescu, R.F., Jordans, S., Rehders, M., Avena, M., Oswald, J., Lerchl, A., Saftig, P., and Brix, K. (2011). Cathepsin K deficiency in mice induces structural and metabolic changes in the central nervous system that are associated with learning and memory deficits. BMC Neurosci., 12.","DOI":"10.1186\/1471-2202-12-74"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"175","DOI":"10.1210\/endo-102-1-175","article-title":"Acute Effects of Thyroid-Stimulating Hormone on Cultured Thyroid Cell Morphology","volume":"102","author":"Rapoport","year":"1978","journal-title":"Endocrinology"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"667","DOI":"10.1152\/physrev.1992.72.3.667","article-title":"Physiological and pathological regulation of thyroid cell proliferation and differentiation by thyrotropin and other factors","volume":"72","author":"Dumont","year":"1992","journal-title":"Physiol. Rev."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"685228","DOI":"10.3389\/fendo.2021.685228","article-title":"The Role of Primary Cilia in Thyroid Cancer: From Basic Research to Clinical Applications","volume":"12","author":"Ma","year":"2021","journal-title":"Front. Endocrinol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"26038","DOI":"10.1016\/S0021-9258(18)35713-2","article-title":"Sequence analysis, tissue distribution, and expression of rat cathepsin S","volume":"267","author":"Petanceska","year":"1992","journal-title":"J. Biol. Chem."},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Al-Hashimi, A., Venugopalan, V., Rehders, M., Sereesongsaeng, N., Hein, Z., Springer, S., Weber, H., F\u00fchrer, D., Bogyo, M., and Scott, C. (2020). Procathepsin V is Secreted in a TSH Regulated Manner from Human Thyroid Epithelial Cells and Is Accessible to an Activity-Based Probe. Int. J. Mol. Sci., 21.","DOI":"10.3390\/ijms21239140"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"7893","DOI":"10.1074\/jbc.M610107200","article-title":"Mechanism of Elastinoysis and Inhibition by Macromolecular inhibitors","volume":"282","author":"Novinec","year":"2007","journal-title":"J. Biol. Chem."},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Qatato, M., Venugopalan, V., Al-Hashimi, A., Rehders, M., Valentine, A.D., Hein, Z., Dallto, U., Springer, S., and Brix, K. (2021). Trace Amine-Associated Receptor 1 Trafficking to Cilia of Thyroid Epithelial Cells. Cells, 10.","DOI":"10.3390\/cells10061518"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"30","DOI":"10.1159\/000434717","article-title":"Trace Amine-Associated Receptor 1 Localization at the Apical Plasma Membrane Domain of Fisher Rat Thyroid Epithelial Cells is Confined to Cilia","volume":"4","author":"Szumska","year":"2015","journal-title":"Eur. Thyroid. J."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/24\/11\/9292\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T19:42:29Z","timestamp":1760125349000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/24\/11\/9292"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,5,26]]},"references-count":32,"journal-issue":{"issue":"11","published-online":{"date-parts":[[2023,6]]}},"alternative-id":["ijms24119292"],"URL":"https:\/\/doi.org\/10.3390\/ijms24119292","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2023,5,26]]}}}