169 Atrial natriuretic factor and C-type natriuretic peptide induce retraction of human thyrocytes in monolayer culture via guanylyl cyclase receptors D F Sellitti1, C Lagranha1,2, G Perrella3, F Curcio3 and S Q Doi1 1Department of Medicine, Divisions of Endocrinology and Nephrology, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, A3060, Bethesda, MD 20814–4799, USA 2Department of Biophysics and Physiology, Division of Endocrinology, University of Sao Paulo, Sao Paulo, Brazil 3Dipartimento di Patologia e Medicina Sperimentale e Clinica, University of Udine, Udine, 33100, Italy (Requests for offprints should be addressed to D F Sellitti) Abstract The natriuretic peptides signal through three receptor increases in retracted cell number were induced by ANF subtypes, of which two (NPR-A and NPR-B) are and CNP, but not the NPR-C-specific ring-deleted ANF membrane-bound guanylyl cyclases for which the princi- analog, C-ANF(4–23), during a 15-day treatment. All pal ligands are respectively atrial natriuretic factor (ANF) three natriuretic peptides, however, induced a small (15– and C-type natriuretic peptide (CNP). In the human 20%) but significant (P<0·001) increase in DNA content thyroid cell, a third receptor, NPR-C, has been implicated per well. The stable analog of cGMP, 8-bromo-cGMP in the regulation of thyroglobulin, but functional roles for (8-BrcGMP; 1 mM), also increased the number of NPR-A and NPR-B have not yet been defined. In the retracted HTU-5 cells, and was equipotent with the present study we used RT-PCR to identify transcripts of cAMP analog, 8-BrcAMP, in this effect. The cGMP- all three receptor subtypes, both in human thyroid and in dependent protein kinase inhibitor, KT5823, however, HTU-5 cells, a long-term culture of thyroid-derived cells. had no significant effect on the ANF-induced increase in Both ANF and CNP induced a twofold increase in numbers of retracted cells. These results suggest that the intracellular cGMP content in HTU-5 cells. Morphologic actions of NPR-A and NPR-B, functional receptors in changes (a significant increase in cells of the retracted the human thyroid cell, may in part be mediated by phenotype) were observed in ANF- and CNP-treated cGMP-induced alterations in the cytoskeleton. cells within 3 and 5 h of treatment respectively. Significant Journal of Endocrinology (2002) 173, 169–176 Introduction (Anand-Srivastava & Trachte 1993, Murthy & Makhlouf 1999, Sellitti et al. 2001). ANF and CNP are the principal The natriuretic peptides (atrial natriuretic factor (ANF), ligands for NPR-A and NPR-B respectively (Lowe et al. brain natriuretic peptide (BNP), and C-type natriuretic 1989, Schultz et al. 1989). NPR-C, however, is relatively peptide (CNP) (Levin et al. 1998)) have been proposed as less selective as to natriuretic peptide structure, and binds a class of endocrine/autocrine regulatory factors in the both ANF and CNP with similar affinity (Fuller et al. mammalian thyroid gland (Hughes & Sellitti 1991, Eggo 1988). NPR-C was the first natriuretic peptide receptor to & Sheppard 1994, Bidey et al. 1999) on the basis of be associated with the regulation of a specific thyroid the co-expression of both natriuretic peptides and their function, following the observation that ANF suppresses receptors (NPRs) in thyroid follicular cells of several thyroglobulin production in human thyrocyte cultures species (Sellitti & Hughes 1990, Tseng et al. 1990, Hughes (Tseng et al. 1990). Furthermore, it was established that & Sellitti 1991, Sellitti et al. 1992, Sellitti & Doi 1994, the thyroglobulin suppression correlated closely with an 1999). Two of the three subtypes of NPR, NPR-A and inhibition of intracellular cAMP production by ANF. NPR-B, are membrane guanylyl cyclases that induce To date, however, no specific function has been ident- intracellular cGMP formation upon binding natriuretic ified specifically with either the NPR-A or NPR-B peptide (Chinkers et al. 1989, Koller et al. 1991, Garbers receptor in thyroid cells, despite several studies demon- & Lowe 1994). The other, NPR-C, is coupled to strating their presence in thyrocytes of rat and porcine adenylyl cyclase in many cell systems, but the mechanism origin (Hughes & Sellitti 1991, Sellitti & Doi 1999). linking this receptor to cAMP remains controversial Nonetheless, previous reports linking cGMP generation to Journal of Endocrinology (2002) 173, 169–176 Online version via http://www.endocrinology.org 0022–0795/02/0173–169 2002 Society for Endocrinology Printed in Great Britain Downloaded from Bioscientifica.com at 09/29/2021 07:48:04AM via free access 170 D F SELLITTI and others · ANF and CNP induce thyrocyte retraction specific functions in the thyroid cell would appear to these cells were assessed and compared with human support a role for NPR guanylyl cyclase/cGMP signaling thyroid and heart controls using RT-PCR for the expres- (Bocanera et al. 1997, Rasmussen et al. 1997). To evaluate sion of the thyroid-restricted genes Pax-8, thyrotropin further the role of NPR subtype in thyroid function, we receptor (TSH-R), and thyroglobulin (Tg) as described have studied the expression of the three NPR subtypes in below. Results indicated the partial de-differentiation of human thyroid and in HTU-5 cells, a long-term culture of HTU-5 cells by passage 13, with the loss of TSH-R and human thyrocytes (Curcio et al. 1994). Human thyrocytes Pax-8 expression. Despite the loss of thyroid-specific gene in culture exhibit distinctive morphologic changes in expression, these cells retained the typical morphology of response to hormonal stimulation, including cytoplasmic human thyrocytes in monolayer culture and their capacity retraction, a process in which the cytoplasm of monolayer for retraction, confirming that the cultures remained cells is partially detached from the substrate, leaving only purely thyrocyte in nature, even after several passages. highly arborized processes in contact with the substrate The basic culture medium for these cells was Coon’s and with neighboring cells (Westermark & Porter 1982, F-12 medium (Sigma) modified by the addition of MgCl2 Lamy et al. 1990, Taton et al. 1993, Asakawa et al. 1996). to a final concentration of 0·5 mM, CaCl2 to 1·1 mM This process has been shown to be hormonally sensitive, and glucose to 3·6 mM. The medium (Humed) was also with both thyrotropin and cAMP treatment effecting an supplemented with 5% fetal bovine serum, a five-hormone increase in the retracted phenotype (Taton et al. 1993). In mixture (insulin, somatostatin, hydrocortisone, transferrin the present study, we have exploited the distinctive and glycyl-histidyl-lysine), and bovine hypothalamus and phenotype of these cells to examine the effects of subtype- bovine pituitary (Pel Freez Biologicals, Rogers, AK, USA) specific natriuretic peptides (ANF, CNP, and C-ANF(4– extracts, as previously described (Curcio et al. 1994). 23)) and their intracellular signaling systems, in thyroid cells of human origin. cGMP assay Materials and Methods Cells were plated randomly and evenly and grown to confluence in Humed medium in 24-well plates. At assay, Reagents growth medium was aspirated and culture wells received 0·4 ml minimal essential medium (MEM) containing Rat ANF (ANF(99–126)), CNP-22, and C-ANF(4–23) 1 mM CaCl2, 1 mM Mg2SO4, 1% bovine serum albumin, were purchased from Peninsula Laboratories (Belmont, 0·5 mM isobutylmethylxanthine, and either ANF or CNP CA, USA). Thyroid-stimulating hormone (TSH) from (0–1 mM). After a 20-min incubation at 37 C, medium bovine pituitary (2 IU/mg protein), 8-bromoguanosine was aspirated and wells received 0·5 ml of a 0·1 M HCl 3,5 -cyclic monophosphate (8-BrcGMP) and 8-bromo- solution containing 0·1 mM CaCl , and these samples 2 adenosine 3,5 -cyclic monophosphate (8-BrcAMP) were were stored at -70 C until required for determination obtained from Sigma Chemical, St Louis, MO, USA. of cGMP content by RIA (Biomedical Technologies, KT5823 was obtained from Calbiochem (San Diego, CA, Staughton, MA, USA). All samples were diluted 1:5 USA). Human thyroid, heart and kidney mRNA were in acetate buffer and acetylated before being assayed. purchased from Clontech (Palo Alto, CA, USA). Significant differences among treatment groups were determined using ANOVA followed by Dunnett’s t-test Cell culture with InStat software (GraphPad Software Inc., San Diego, CA, USA). A clonal strain of human thyroid cells (HTU-5) was developed from normal human thyroid tissue and grown using methods previously reported (Curcio et al. 1994). In Retracted cell count brief, normal thyroid tissue from pathological specimens was freed from adherent connective tissue, cut into small HTU-5 cells were plated randomly and evenly and grown pieces, washed in Ca2+- and Mg2+-free Hanks’s balanced to confluence in Humed medium in 24-well plates. After salt solution (HBSS) and digested with a solution consisting treatment (ANF, CNP, C-ANF(4–23), 8-BrcAMP or of 20 U/ml collagenase, 0·75 mg/ml trypsin, and 2% 8-BrcGMP), randomly selected fields from the center heat-inactivated dialyzed chicken serum, in Ca2+- and of each well were observed under low power (10 Mg2+-free HBSS. Following a 2-h digestion, cell suspen- objective) using a phase-contrast microscope (Olympus), sions were seeded onto 100-mm plastic culture dishes in a and the total number of retracted cells in the field was culture medium consisting of a modified F-12 medium as counted. A cell was considered to be retracted if the described earlier (Trusolino et al. 1998), and in which cell body was refractile, with extensive cytoplasmic arbori- secondary cultures were passaged. zation in place of the normal flattened monolayer pheno- In the present study, HTU-5 cells were used between type. Mean counts of retracted cells (retracted cells/field) passages 9 and 16.