Circulating thyroglobulin transcytosed by thyroid cells in complexed with secretory components of its endocytic receptor megalin

After its endocytosis from the colloid, some thyroglobulin (Tg) is transcytosed intact across thyrocytes, accounting in part for its presence in the circulation. We previously showed that megalin (gp330), an endocytic Tg receptor, mediates apical to basolateral Tg transcytosis. Here we investigated whether a portion of megalin remains combined with Tg after its transcytosis, using studies with cultured thyroid cells and in vivo observations. FRTL-5 cells, a rat thyroid cell line, cultured on filters in dual chambers form tight junctions and exhibit features of polarity, with expression of megalin exclusively on the upper (apical) surface. After the addition of unlabeled Tg to the upper chamber and incubation at 37 C, some Tg was transcytosed intact across FRTL-5 cells into the lower chamber. Two antimegalin ectodomain antibodies precipitated transcytosed Tg in fluids collected from the lower chamber. After the addition of Tg to surface-biotinylated FRTL-5 cells, an anti-Tg antibody and the two antimegalin ectodomain antibodies precipitated high molecular mass biotinylated material in fluids collected from the lower chamber, corresponding to much of the megalin ectodomain, as well as smaller amounts of lower molecular mass material. The results indicate that Tg transcytosed across FRTL-5 cells remains complexed with megalin ectodomain components, which we refer to as megalin secretory components. In aminotriazole-treated rats, which develop increased megalin-mediated Tg transcytosis, antimegalin antibodies precipitated some of the Tg in the serum. Tg was also precipitated by antimegalin antibodies in sera from patients with Graves' disease, in which we found increased megalin expression on the apical surface of thyrocytes. In contrast, in thyroidectomized patients with metastatic papillary thyroid carcinoma, in whom Tg is directly secreted by neoplastic thyroid cells into the circulation rather than transcytosed, serum Tg was not precipitated by antimegalin antibodies. The detection of Tg-megalin complexes may help identify the source of serum Tg in patients with thyroid diseases.     

Megalin-mediated transcytosis of thyroglobulin by thyroid cells is a calmodulin-dependent process.

Megalin, a multiligand receptor expressed on the apical surface of thyroid cells, mediates transepithelial transport (transcytosis) of thyroglobulin (Tg) across thyrocytes, resulting in diversion of Tg from the lysosomal pathway and reduction of the extent of thyroid hormone release from internalized Tg molecules. The calcium regulatory protein calmodulin facilitates some forms of transcytosis. Here we investigated the role of calmodulin in megalin-mediated transcytosis of Tg by thyroid cells. For this purpose, we studied the effect of calmodulin antagonists on Tg transcytosis by Fisher rat thyroid cells (FRTL-5), an established, differentiated thyroid cell line. FRTL-5 cells were cultured on permeable filters in the upper chamber of dual chambered devices, with megalin expression exclusively on the upper surface. Unlabeled Tg was added to the upper chamber at 37 degrees C, and transcytosed Tg was detected by enzyme-linked immunosorbent assay (ELISA) in fluids collected 1 hour later from the lower chamber. To study the role of calmodulin in Tg transcytosis, cells were preincubated with one of two calmodulin antagonists, either trifluoperazine or W7. Both antagonists markedly reduced transcytosis of Tg by FRTL-5 cells. These inhibitory effects and those of a monoclonal antimegalin antibody were not additive, indicating that calmodulin acts on the megalin-mediated pathway. Furthermore, trifluoperazine increased the extent of triiodothyronine (T3) release from exogenously added Tg by FRTL-5 cells, indicating that Tg transported in the calmodulin-dependent, megalin-mediated pathway, bypasses the lysosomal pathway.     

Binding of rat thyroglobulin to heparan sulfate proteoglycans.

We previously showed that rat thyroglobulin (Tg) is a heparin-binding protein and that heparin inhibits Tg binding to megalin (gp330), an endocytic Tg receptor found on the apical surface of thyrocytes. Cooperation between cell surface receptors and heparin-like molecules, namely heparan sulfate proteoglycans (HSPGs), can facilitate cell surface binding of some heparin-binding proteins. Based on our previous findings indicating that heparin and megalin-binding sites of rat Tg are functionally related, here we investigated whether rat Tg binds to HSPGs, which are expressed by thyroid cells. We showed in solid phase assays that unlabeled rat Tg binds to a heparan sulfate (HS) preparation in a dose-dependent, saturable manner, with moderately high affinity (Kd approximately 19 nM, Ki approximately 25 nM). Binding was inhibited by heparin and by HS itself. We then studied the role of HSPGs in Tg binding to FRTL-5 cells, a differentiated Fisher rat thyroid cell line. As previously reported, after incubation of FRTL-5 cells with unlabeled rat Tg at 4 degrees C, heparin released virtually all the cell-bound Tg. Co-incubation of Tg with HS or with a preparation of HSPGs resulted in a reduction of binding by 35%-40%. When FRTL-5 cells were preincubated with heparitinase or heparinase I, which released 20%-30% of cell surface HSPGs, Tg binding was reduced to a similar extent. An antibody against a Tg heparin-binding site functionally related to a major megalin-binding site virtually abolished Tg binding to HS and to FRTL-5 cells, supporting the hypothesis that combined interactions of Tg with HSPGs and with megalin are involved in Tg binding to rat thyroid cells.     

Phosphoinositide 3-kinase inhibits megalin-mediated transcytosis of thyroglobulin across thyroid epithelial cells at a post-sorting level

BACKGROUND: Phosphoinositide 3-kinase (PI3-K) is implicated in various cellular processes involving signaling, including intracellular trafficking. PI3-K has been shown to play a part in both receptor- and non-receptor-mediated transcytosis across cultured kidney cells and undifferentiated thyroid cells. OBJECTIVE: To investigate the role of PI3-K in transcytosis of thyroglobulin (Tg) across differentiated cultured Fisher rat thyroid cells (FRTL-5 cells) - a process known to be mediated by megalin, a member of the low-density lipoprotein receptor family. DESIGN: We studied the effect of the microbial product wortmannin, a specific inhibitor of PI3-K, on transcytosis of Tg across FRTL-5 cells. METHODS: Transcytosis experiments were performed using FRTL-5 cells cultured as tight layers on filters in the upper chamber of dual chambered devices, with megalin expression exclusively on the upper cell surface. Tg was added to the upper chamber and cells were incubated at 37 degrees C. Transcytosed Tg was measured in fluids collected from the lower chamber. To study the role of PI3-K, cells were pre-incubated with wortmannin. RESULTS: Pre-incubation of FRTL-5 cells with wortmannin did not affect Tg binding and uptake, but resulted in a considerable increase in Tg transcytosis (by 40-75%, depending on the concentration of wortmannin), suggesting that PI3-K exerts an inhibitory effect on Tg transcytosis. In experiments in which a monoclonal antibody against megalin was used to reduce Tg transcytosis, pre-incubation with wortmannin did not increase Tg transcytosis from its reduced levels, indicating that PI3-K is involved in the megalin-mediated pathway. Wortmannin did not affect the extent of release of tri-iodothyronine from exogenously added Tg by FRTL-5 cells, which was used as a measure of Tg degradation in the lysosomal pathway, indicating that the effect of PI3-K on transcytosis occurs after diversion of Tg from the lysosomal pathway. CONCLUSIONS: PI3-K exerts an inhibitory role on megalin-mediated Tg transcytosis across cultured thyroid cells. PI3-K action takes place at a post-sorting level, after Tg bypassing of the lysosomal pathway.     

Megalin in thyroid physiology and pathology.

Megalin, a member of the low density lipoprotein endocytic receptor family, is expressed on the apical surface of thyroid epithelial cells, directly facing the follicle lumen, where colloid is stored in high concentrations. Studies in vivo and with cultured thyroid cells have provided evidence that megalin expression on thyroid cells is TSH-dependent. Thyroglobulin (Tg), the major protein component of the colloid and the precursor of thyroid hormones, binds to megalin with high affinity and megalin mediates in part its uptake by thyrocytes. Tg internalized by megalin avoids the lysosomal pathway and is delivered by transepithelial transport (transcytosis) to the basolateral membrane of thyrocytes, from which it is released into the bloodstream. This process competes with pathways leading to thyroid hormone release from Tg molecules, which occurs following internalization of Tg molecules from the colloid by other means of uptake (fluid phase endocytosis or endocytosis mediated by low affinity receptors) that result in proteolytic cleavage in the lyosomes. During transcytosis of Tg, a portion of megalin (secretory component) remains complexed with Tg and enters the circulation, where its detection may serve as a tool to identify the origin of serum Tg in patients with thyroid diseases. Tg endocytosis via megalin is facilitated by the interaction of Tg with cell surface heparan sulfate proteoglycans, which occurs via a carboxyl terminal heparin binding site of Tg functionally related with a major megalin binding site. Although autoantibodies against megalin can be found in the serum of approximately 50% of patients with autoimmune thyroiditis, a role of megalin in this and other thyroid diseases remains to be established.     

ClC-5 Does Not Affect Megalin Expression and Function in the Thyroid.

Megalin is an endocytic receptor responsible for thyroglobulin (Tg) transcytosis, a process that favors hormone release. Accordingly, megalin KO mice have primary hypothyroidism. In the kidney, megalin expression is reduced when the gene encoding the chloride transporter ClC-5 is mutated. We investigated whether megalin expression and function in the thyroid are affected by ClC-5 using a ClC-5 KO mouse model. By Western blotting, ClC-5 was found in thyroid tissue extracts of WT, but not of ClC-5 KO mice. In addition, ClC-5 was found to be expressed by cultured thyroid cells (FRTL-5). The thyroid size, weight, and histology were similar in ClC- 5 KO and WT mice, as were the amounts of megalin in thyroid extracts. Accordingly, serum Tg, a measure of megalin-mediated transcytosis, was similar in WT and ClC-5 KO mice, suggesting that megalin function was unaffected. Thus, unlike in megalin KO mice, in ClC-5 KO mice thyroid function was unchanged, as indicated by the normal serum FT4 and TSH. We concluded that in the thyroid, unlike in the kidney, ClC-5 does not affect megalin expression and function, suggesting that megalin is differentially regulated in these two organs.     

Targeting of thyroglobulin to transcytosis following megalin-mediated endocytosis: evidence for a preferential pH-independent pathway

TG internalized from the colloid by megalin, bypasses the lysosomal pathway and is transported across thyrocytes by transcytosis. Although most of the intracellular mechanisms responsible for targeting of ligands to transcytosis are unknown, for certain ligands a role of lysosomal pH has been established. Thus, ligands that undergo lysosomal degradation dissociate from their receptors due to the low pH of endosomes, whereas certain ligands that undergo transcytosis fail to dissociate because they bind to their receptors at acidic pH. Here we studied the role of pH in TG transcytosis. We first investigated the effect of pH on megalin binding to TG in solid phase assays and found that, although megalin bound to TG at various pH values (ranging from 4-8), optimal binding was seen at acidic pH (ranging from 4.5-6). We then studied the effect of chloroquine (CQ) and ammonium chloride (AC), which increase endosomal pH, on transcytosis of TG across Fisher rat thyroid (FRTL-5 cells). Transcytosis assays were performed using FRTL-5 cells cultured on filters in dual chambered devices, with megalin expression only on the upper surface of the layers. TG was added to the upper chamber and transcytosed TG was measured in fluids collected from the lower chamber after incubation at 37 C. Treatment of FRTL-5 cells with CQ or AC did not affect binding and uptake of TG, but it did reduce T3 release from exogenously added TG, used as a measure of TG degradation in the lysosomal pathway. Treatment with CQ or AC resulted in an increase of transcytosis of TG across FRTL-5 cells, but only to a minimal extent (15-20%). The effects of CQ or AC and those of a megalin competitor (the monoclonal antibody 1H2, which reduced transcytosis) were not additive, suggesting that CQ and AC act on the megalin-mediated pathway. In conclusion, because TG binding to megalin is greatest at acidic pH, it is possible that TG does not dissociate from megalin in the lysosomal pathway. However, the pH-dependence of TG binding to megalin does not account for much of transcytosis, which probably occurs largely because of other mechanisms of targeting.     

Thyroid dysfunction in megalin deficient mice

Megalin mediates transcytosis of thyroglobulin (Tg), the thyroid hormone precursor, resulting in its passage into the bloodstream. The process involves especially hormone-poor Tg, which may favour hormone secretion by preventing competition with hormone-rich Tg for proteolytic degradation. To gain more insight into the role of megalin, here we studied thyroid function and histology in megalin deficient mice compared with WT mice. As expected from the knowledge that megalin mediates Tg transcytosis, serum Tg levels were significantly reduced in homozygous (megalin-/-) mice, which, more importantly, were found to be hypothyroid, as demonstrated by significantly reduced serum free thyroxine and significantly increased serum thyroid stimulating hormone (TSH) levels. In heterozygous (megalin+/-) mice, in which megalin expression was normal, thyroid function was unaffected. Although the serological phenotype in megalin-/- mice was not associated with histological alterations or goiter, our results support a major role of megalin in thyroid hormone secretion.     

Binding of heparin to human thyroglobulin (Tg) involves multiple binding sites including a region corresponding to a binding site of rat Tg

OBJECTIVE: Binding of thyroglobulin (Tg) to heparin allows efficient Tg interaction with its endocytic receptor, megalin. Rat Tg (rTg) binds to heparin using an exposed carboxyl terminal region (RELPSRRLKRPLPVK, Arg2489-Lys2503) rich in positively charged residues which is, however, not entirely conserved in human Tg (hTg) (Arg2489-Glu2503, REPPARALKRSLWVE). Here, we investigated whether and how this difference affects binding of heparin. DESIGN: To compare binding of heparin to rTg and hTg. To investigate the role of the sequence 2489-2503 using a peptide-based approach. METHODS: Binding of biotin-labeled heparin to rTg, hTg and to Tg peptides was measured in solid phase assays. RESULTS: Heparin bound to rTg with moderately high affinity (K(d): 34.2 nmol/l, K(i): 37.6 nmol/l) and to hTg with lower affinity (K(d): 118 nmol/l, K(i): 480 nmol/l) and to a lower extent. Binding was dose-dependent and saturable, and was reduced by several specific competitors (Tg itself, unlabeled heparin, lactoferrin). Heparin bound to synthetic peptides corresponding to the rat (rTgP) and to the human (hTgP) Tg sequence 2489-2503. Heparin bound to rTgP to a greater extent and with greater affinity than to hTgP. An antibody against hTgP reduced binding of heparin to intact hTg by 30%, suggesting that in hTg this region is, in part, involved in heparin binding, but also that other regions account for most of the binding. Starting from the sequence of rTgP, we designed 6 synthetic 'mutant' peptides by replacing one amino acid residue of rTgP with the corresponding residue of the sequence of hTgP. Heparin bound to 5 of 6 mutant peptides to a lower extent and with lower affinity than to rTgP. CONCLUSIONS: In spite of a reduced binding ability of the sequence 2489-2503, hTg binds to heparin, in part, using alternative, as yet unidentified, binding sites. Substitution of both positive and neutral residues within the sequence 2489-2503 reduced heparin-binding, suggesting that not only charge, but also sequence and/or conformation, may account for the heparin-binding ability of this region of Tg.     

Sortilin is a putative postendocytic receptor of thyroglobulin

The Vps10p family member sortilin is involved in various cell processes, including protein trafficking. Here we found that sortilin is expressed in thyroid epithelial cells (thyrocytes) in a TSH-dependent manner, that the hormone precursor thyroglobulin (Tg) is a high-affinity sortilin ligand, and that binding to sortilin occurs after Tg endocytosis, resulting in Tg recycling. Sortilin was found to be expressed intracellularly in thyrocytes, as observed in mouse, human, and rat thyroid as well as in FRTL-5 cells. Sortilin expression was demonstrated to be TSH dependent, both in FRTL-5 cells and in mice treated with methimazole and perchlorate. Plasmon resonance binding assays showed that Tg binds to sortilin in a concentration-dependent manner and with high affinity, with Kd values that paralleled the hormone content of Tg. In addition, we found that Tg and sortilin interact in vivo and in cultured cells, as observed by immunoprecipitation, in mouse thyroid extracts and in COS-7 cells transiently cotransfected with sortilin and Tg. After incubation of FRTL-5 cells with exogenous, labeled Tg, sortilin and Tg interacted intracellularly, presumably within the endocytic pathway, as observed by immunofluorescence and immunoelectron microscopy, the latter technique showing some degree of Tg recycling. This was confirmed in FRTL-5 cells in which Tg recycling was reduced by silencing of the sortilin gene and in CHO cells transfected with sortilin in which recycling was increased. Our findings provide a novel pathway of Tg trafficking and a novel function of sortilin in the thyroid gland, the functional impact of which remains to be established.     

Failure to use measurement of megalin secretory components complexed with serum thyroglobulin as a tool to identify metastases after surgery in papillary thyroid cancer

When thyroid follicles are intact, some colloidal thyroglobulin (Tg) reaches the circulation by megalin-mediated transcytosis and is to various extents complexed with megalin secretory components. In contrast, in papillary thyroid cancer (PTC), serum Tg is not complexed with megalin because it is directly secreted by tumor cells. Here we attempted to use measurement of megalin secretory components to distinguish PTC patients with thyroid remnant plus metastases from those with thyroid remnant only, after thyroidectomy and before 131I ablation. Tg values in anti-Tg antibodies (TgAb)-free sera from 5 PTC patients with thyroid remnant plus metastases and 12 PTC patients with thyroid remnant only were measured following pre-adsorption with uncoupled protein A beads or with protein A beads coupled with antimegalin antibodies. The degree of Tg pre-adsorption with antimegalin antibodies was minimal, with no substantial differences between the two groups. Thus, we concluded that measurement of megalin secretory components is unlikely to be useful to identify the origin of serum Tg in PTC patients after thyroidectomy.     

Binding, uptake, and degradation of internalized thyroglobulin in cultured thyroid and non-thyroid cells

Thyroid hormone release requires degradation of thyroglobulin (Tg) by thyroid epithelial cells, which occurs mainly in the lysosomal pathway following Tg endocytosis. Non-specific fluid-phase endocytosis is thought to be the main route of Tg uptake leading to degradation, whereas receptor- mediated endocytosis is believed to lead to post-endocytic pathways other than degradation. To gain more insights into these issues, we investigated handling of Tg by various cell types. Tg bound similarly to thyroid (FRTL-5, FRT) and non-thyroid (COS-7, IRPT) cells, indicating the presence of membrane-binding sites, presumably receptors, in both cell types. Tg was internalized and degraded by all cells and degradation paralleled uptake, with the exception of FRTL- 5 cells, in which a lower proportion of Tg was degraded, suggesting that in FRTL-5 cells mechanisms that target Tg to the various post-endocytic pathways (either receptors or postreceptorial factors) are differently represented. Immunoelectronmicroscopy showed a common path of endocytosis in FRTL-5, COS-7, and IRPT cells, namely the formation of pseudopods engulfing Tg, followed by internalization and accumulation of Tg in cytoplasmic vesicles and lysosomes. The fastest rate was observed in COS-7 cells, probably reflecting a lower impact of endocytic receptors. Our findings suggest that Tg uptake and degradation are not thyroid-specific, that Tg binding sites exist in different cell types, and that uptake and/or degradation are differently regulated in differentiated thyroid cells, presumably because of a different impact of endocytic receptors or post-endocytic mechanisms, which are probably responsible for the regulation of hormone release.     

Mechanisms of bradykinin-induced glucagon release in clonal alpha-cells In-R1-G9: involvement of Ca(2+)-dependent and -independent pathways

Recent studies suggest striking similarities between polarized protein sorting in thyrocytes and MDCK epithelial cells, including apical trafficking of thyroglobulin (Tg), thyroid peroxidase, and aminopeptidase N; as well as basolateral targeting of heparan sulfate proteoglycans, thrombospondin 1 (TSP1), type 1 5'-deiodinase, sodium-potassium ATPase, and the thyrotropin receptor. In this report, we have firstly expressed in stably transfected MDCK II cells a range of truncation mutants lacking up to 78% of the C-terminus of TSP1; these studies indicate that the N-terminal region containing the heparin binding domain is sufficient for basolateral targeting of TSP1. Secondly, we have stably transfected MDCK II cells with both Tg and sodium-iodide symporter (NIS) cDNAs, obtaining clones that simultaneously express both thyroid-specific proteins at the apical and basolateral cell surfaces, respectively. These studies represent promising early steps towards designing artificial thyrocytes by thyroid gene transfer into MDCK cells.     

Impaired thyroglobulin (Tg) secretion by FRTL-5 cells transfected with soluble receptor associated protein (RAP): evidence for a role of RAP in the Tg biosynthetic pathway

Secretion of thyroglobulin (Tg) by thyrocytes requires several endoplasmic reticulum (ER)-resident molecular chaperones. The receptor-associated protein (RAP), a known molecular chaperone, binds to Tg in thyroid cells shortly after biosynthesis. Here we investigated whether RAP is involved in Tg secretion by FRTL-5 cells. For this purpose, we studied Tg secretion by FRTL-5 cells transfected with a soluble RAP chimera, as a mean for interfering with endogenous RAP. We used a RAP-human IgG Fc (RAP-Ig) chimeric cDNA, which was designed in order to exclude the ER retention sequence of RAP and to allow generation of a secreted form of RAP. FRTL-5 cells were transiently transfected with the RAP-Ig cDNA or, as control, with a CD8-Ig cDNA. Media were collected at 24, 48 and 72 h after transfection. Secretion of fusion proteins and of Tg in the media was measured by ELISA. As expected, under standard culture conditions, RAP was not secreted into the media by FRTL-5 cells, even though it could be detected by Western blotting in cell extracts. In transfection experiments, fusion proteins were present in the media of FRTL-5 cells transfected with either RAP-Ig or CD8-Ig, indicating that transfection was successful. Although Tg was found in the media of FRTL-5 cells transfected with either CD8-Ig or RAP-Ig, a lower amount was found in cells transfected with RAP-Ig. Therefore, we concluded that RAP is involved in Tg secretion by FRTL-5 cells suggesting that RAP may function as a Tg molecular chaperone.     

Adhesion molecule expression by the FRTL-5 rat thyroid cell line.

We have examined the expression and function of rat CD54, a homologue of human intercellular adhesion molecule-1 (ICAM-1), by the continuously growing rat thyroid cell line FRTL-5. Approximately 10% of FRTL-5 cells express CD54 under basal conditions and this is not influenced by thyrotrophin. Expression of CD54 is increased by cytokines (gamma-interferon, tumour necrosis factor, interleukin-1) and by an activator of C-kinase, phorbol 12-myristate 13-acetate. Blocking ICAM-1 with a monoclonal antibody directed against this molecule significantly (P less than 0.01) reduced the binding of splenic lymphocytes to FRTL-5 cells but inhibition was consistently greater (P less than 0.01) in the presence of antibodies against a rat homologue of lymphocyte function-associated antigen-1, the receptor on T cells for ICAM-1. In no case was complete blocking of cluster formation observed. These results show that a pure line of rat thyroid cells can express an ICAM-1 homologue and this is directly enhanced by cytokines. Expression of this homologue is partially responsible for lymphocyte adhesion to thyroid cells, which is likely to be a major event in T cell recognition of thyroid antigens in autoimmune thyroiditis.     

Control of thyroglobulin secretion from the rat thyroid gland

Serum thyroglobulin (Tg), measured by radioimmunoassay, was high in 6-propylthiouracil (PTU)-treated rats but low in thyroxine (T4)-treated animals compared with euthyroid controls. Thyroid-stimulating hormone (TSH) stimulated Tg release in vitro from enzymatically dispersed normal rat thyroid cells in a dose-dependent manner. Thyroid cells prepared from T4-treated animals behaved similarly to cells from control rats, whereas in vitro basal release of Tg from thyroid cells prepared from PTU-treated animals was high and the response to TSH was lost. Our data confirm the TSH dependency of Tg release in vivo and in vitro and our system provides a means of studying the control of Tg secretion in vitro.     

Effect of lithium on function and growth of thyroid cells in vitro.

Lithium has been reported to alter thyroid function and cause goiter in some patients. To explain the mechanism of lithium action in the thyroid gland, we studied the effect of lithium on thyroid function and cell growth in FRTL-5 rat thyroid cells and on de novo thyroid hormone formation in primary cultures of porcine thyroid follicles. TSH-induced iodide uptake was suppressed at 2 mM lithium in both FRTL-5 cells and porcine follicles. In porcine thyroid follicles, iodide uptake stimulated by 8-bromo-cAMP, iodine organification, and de novo thyroid hormone formation were also reduced by lithium; however, 2 mM lithium did not inhibit TSH-induced cAMP production. In FRTL-5 cells, lithium also inhibited forskolin-stimulated iodide uptake. These results suggested that lithium exerts its effect at a step involving cAMP signal transduction rather than inhibiting cAMP production. In both FRTL-5 thyroid cells and porcine follicles, lithium enhanced cell growth in basal states (lacking TSH) and with TSH treatment. In porcine thyroid cells, the protein kinase C activator, tetradecanoyl phorbol-13-acetate, increased cell growth, and lithium had an additive effect with tetradecanoyl phorbol-13-acetate on cell growth. To examine the possibility that the action of lithium was mediated by the protein kinase C pathway, porcine cells were incubated with lithium and H7, a selective protein kinase C inhibitor. Lithium-induced cell growth was suppressed to the basal level by H7. These results suggest that lithium exerts its growth-promoting effect through the protein kinase C system.