Ret-mediated mitogenesis requires Src kinase activity.

The proto-oncogene RET encodes a transmembrane growth neurotrophic receptor with tyrosine kinase (TK) activity. RET mutations are associated with several human neoplastic and nonneoplastic diseases, including thyroid papillary carcinoma, multiple endocrine neoplasia type 2 syndromes, and Hirschsprung's disease. Activation of receptor TKs results in the binding and activation of downstream signaling proteins, among which are nonreceptor TKs of the Src family. To test the involvement of c-Src in Ret-mediated signaling, we measured the levels of c-Src activity in NIH3T3 cells coexpressing Ret and the accessory GFR alpha-1 receptor or an epidermal growth factor receptor/Ret chimeric receptor when the cells were stimulated by glial cell line-derived neurotrophic factor or epidermal growth factor, respectively. Ret stimulation resulted in the activation of c-Src. We also measured the levels of Src kinase activity in cell lines expressing isoforms of the Ret receptor activated by different mutations. These cells showed higher Src kinase activity than the normal counterpart. Furthermore, we show that Ret is able to associate with the SH2 domain of Src in a phosphotyrosine-dependent fashion. Microinjection of a kinase inactive mutant of c-Src blocked Ret-mediated mitogenic effect. These experiments demonstrate that activated Ret is able to bind and stimulate c-Src kinase and that Src activation is essential for the mitogenic activity of Ret.

Expression of the RET/PTC1 oncogene impairs the activity of TTF-1 and Pax-8 thyroid transcription factors.

The most frequent genetic alterations described thus far in human papillary thyroid carcinomas are somatic rearrangements of the RET proto-oncogene, which generate the chimeric RET/PTC oncogenes. We recently found that the expression of the RET/PTC1 oncogene blocked the expression of the thyroid-differentiated phenotype in rat thyroid epithelial cell line PC CI 3 (PC). Here, we show that this block occurs at a transcriptional level; indeed, the thyroid-specific thyroglobulin and thyroperoxidase gene promoters were inactive in PC-PTC cells. Specific transcription factors, namely, TTF-1 and Pax-8, regulate the expression of differentiated functions in thyroid cells. Here, we show that Pax-8 is expressed at reduced levels in PC-PTC cells and that its adoptive overexpression is unable to restore the activity of target promoters. In contrast, TTF-1 expression is unaltered in PC-PTC cells; however, by using a synthetic promoter that contains its specific target sequence, we demonstrate that TTF-1 is inactive in PC-PTC cells. We conclude that the RET/PTC1 oncogene alters the expression of the thyroid-differentiated phenotype by at least two different mechanisms, ie., down-regulation of Pax-8 protein and mRNA expression and impaired function of TTF-1 and Pax-8, which occurs at a posttranslational level.

Only the substitution of methionine 918 with a threonine and not with other residues activates RET transforming potential.

Specific point-mutations of the RET receptor tyrosine kinase protooncogene are responsible for the inheritance of multiple endocrine neoplasia type 2A (MEN2A) and 2B (MEN2B), and familial medullary thyroid carcinoma (FMTC). MEN2B is caused by the substitution of methionine 918 by a threonine in the tyrosine kinase (TK) domain of RET. This mutation converts RET into a dominant transforming oncogene. We have substituted Met918 with four different residues and found that RET acquired transforming activity only when Met918 was substituted with a threonine. However, also when serine and valine, but not leucine or phenylalanine, were inserted in position 918, the RET TK function was activated and induced, especially in the case of the RET(918Ser), immmediate-early response genes. We conclude that the preservation of Met918 is critical for the control of RET kinase. However, only when a threonine residue is present in position 918, does RET efficiently couple with a transforming pathway.

The different RET-activating capability of mutations of cysteine 620 or cysteine 634 correlates with the multiple endocrine neoplasia type 2 disease phenotype.

Distinct point mutations of RET, a tyrosine-kinase receptor encoding gene, are responsible for the inheritance of multiple endocrine neoplasia type 2 syndromes (MEN2A and MEN2B) and familial medullary thyroid carcinoma (FMTC). In particular, MEN2A is a more complex and aggressive disease than FMTC, being characterized by pheochromocytomas and parathyroid alterations, in addition to medullary thyroid carcinomas. The mutations associated with MEN2A and FMTC affect one of five cysteine residues mapping in the extracellular domain of the Ret protein. However, recent studies have indicated that MEN2A and FMTC disease phenotypes correlate with the position of mutations in RET. Mutations of Cys-634 are more frequent in families with MEN2A, whereas Cys-620 mutations are very rarely found in MEN2A patients and, in contrast, are frequently found in FMTC patients. We have reported previously that mutations of Cys-634 constitutively activate the RET transforming potential by causing a disulfide bridge-mediated homodimerization. Here, we report that the mutation Cys-620 --> Tyr is able to cause a constitutive dimerization of Ret, with consequent activation of its kinase and transforming activities, to a lower extent than mutation of Cys-634. We suggest that the difference in ability to activate RET shown by mutations associated with FMTC and MEN2A represents the molecular basis of the phenotypic diversity between the two syndromes.

Loss of thyrotropin regulation and transforming growth factor beta-induced growth arrest in erbB-2 overexpressing rat thyroid cells.

Amplification of erbB-2 gene and overexpression of gp185erbB-2 gene product is found in approximately one-third of primary human breast and ovarian cancer. Overexpression of gp185erbB-2 was recently found in human papillary thyroid carcinomas, but not in thyroid follicular carcinomas or adenomas. The erbB-2 gene encodes a cell surface growth factor receptor with intrinsic tyrosine kinase activity. Wild type human erbB-2 has been shown to act as a potent oncogene when overexpressed in mouse fibroblasts. To test whether overexpression of normal human erbB-2 gene can transform epithelial differentiated rat thyroid cells, these cells were infected with a recombinant retroviral expression vector containing the erbB-2 protooncogene. Rat thyroid cells expressing high levels of gp185erbB-2 do not display a fully transformed and tumorigenic phenotype. However, the isolated cell clones that overexpress gp185erbB-2, show changes in their growth properties if compared to normal thyroid cells, since they can grow in absence of thyrotropin, the main growth factor controlling thyroid cell proliferation in vitro, and do not respond to the growth inhibitory effect of transforming growth factor beta.

Thyroid-stimulating hormone-regulated growth and cell cycle distribution of thyroid cells involve type I isozyme of cyclic AMP-dependent protein kinase.

Optimal growth and differentiation of normal rat thyroid FRTL5 cells depend strictly on the presence of thyroid-stimulating hormone (TSH). FRTL5 cells deprived of TSH cease dividing and become quiescent. Addition of TSH to quiescent cells, which activates the cyclic AMP-mediated pathway, is sufficient to stimulate cell entry into S phase of the cell cycle. We have previously shown that the differential expression of the two isozymes, type I and type II, of the cyclic AMP-dependent protein kinase (PKA) correlates with cell growth and differentiation of several rodent and human cell lines. We have studied the role of PKA in the TSH-regulated growth and cell cycle distribution of FRTL5 cells. Upon addition of TSH to FRTL5 cells deprived of hormone, a rapid induction of RI alpha mRNA species occurred within 30 min after treatment, reaching the levels of proliferating FRTL5 cells at 12 h. RII alpha mRNA levels slightly increased after TSH addition, whereas C alpha mRNA levels did not show major changes. Photoaffinity labeling of PKA receptor proteins showed that addition of TSH to quiescent FRTL5 cells induced a progressive increase in RI alpha levels starting at 6 h after stimulation, whereas RII alpha receptor levels increased only slightly. When FRTL5 cells were treated with an antisense oligodeoxynucleotide targeted against the RI alpha regulatory subunit, their growth was arrested, whereas an antisense against the RII alpha regulatory subunit produced only a mild growth inhibition. Moreover, exposure to the antisense RI alpha oligomer resulted in accumulation of cells in the G0-G1 compartment, as during TSH deprivation.(ABSTRACT TRUNCATED AT 250 WORDS)

TGF beta inhibits rat thyroid cell proliferation without alterations in the expression of TSH-induced cell cycle-related genes.

Transforming growth factor beta (TGF beta) is a secreted polypeptide factor that is thought to play a major role in the regulation of proliferation of many cell types and various differentiation processes. TGF beta acts on thyroid cells by inhibiting cell proliferation and expression of differentiation markers, such as thyroglobulin production and iodide uptake. Exponentially growing thyroid cells cultures accumulate mostly in G0/G1 after exposure to TGF beta for 48 hours. TGF beta inhibits the TSH induced transition of quiescent thyroid cell from the G0 to the S phase. These effects on the thyroid cell growth, however, are not mediated by changes in the TSH-induced cell cycle-related genes expression; both immediate early and progression genes expression is unaffected by the TGF beta treatment.

TSH is able to induce cell cycle-related gene expression in rat thyroid cell.

Rat thyroid cells in culture (FRTL-5 strain) require thyrotropic hormone (TSH) for growth. TSH alone in serum free medium is able to induce DNA synthesis of FRTL-5 cells. DNA synthesis occurs 18-20 hours following TSH stimulation of quiescent cells. Here we demonstrate that two sets of genes, related to the entry of cells in the S phase, are induced by TSH: 1) immediate early genes, such as c-jun and a gene coding for a zinc-finger protein Xrox 20/Egr2, both having a pattern of expression similar to the c-fos oncogene; 2) early delayed genes such as ornithine decarboxylase (ODC), 2F-1, a gene that shows a strong similarity in aminoacid sequence to a mitochondrial ADP/ATP carrier, and the asparagine synthetase gene (TS11). Furthermore, an increased expression of the histone H3 gene, a typical marker of S phase, has been observed in TSH-treated FRTL-5 cells.

Constitutive expression of transforming growth factor alpha does not transform rat thyroid epithelial cells.

To evaluate the role of transforming growth factor alpha (TGF alpha) in transformed rat thyroid epithelial cells, expression and production of TGF alpha were measured in a normal rat thyroid epithelial cell line, FRTL-5, and in the same cells transformed by the Ki-ras oncogene. FRTL-5 cells transformed with Ki-ras exhibit a 5- to 7-fold increase in the levels of TGF alpha-specific mRNA and a 3- to 4-fold enhancement of the amounts of TGF alpha protein in the conditioned medium, as compared with the normal thyroid cells. Although conditioned medium from the Ki-ras transformed FRTL-5 cells or authentic epidermal growth factor or TGF alpha are able to stimulate the anchorage-dependent growth of nontransformed FRTL-5 cells, neither conditioned medium nor the growth factors are able to induce the anchorage-independent growth of these cells in soft agar. FRTL-5 cells were then transfected with an expression vector plasmid containing the human TGF alpha cDNA to directly ascertain if over-expression of this growth factor is able to induce transformation in these cells. The TGF alpha-transfected FRTL-5 clones constitutively produced high amounts of TGF alpha at a level equivalent to or greater than the level found in the conditioned medium from the Ki-ras transformed FRTL-5 clones. Moreover, in contrast to the Ki-ras transformed cells, the TGF alpha transfectants were unable to grow in soft agar, did not form tumors in nude mice, and showed no reduction in the secretion of thyroglobulin. These data demonstrate that unlike Ki-ras, the constitutive expression of biologically active TGF alpha is not entirely sufficient to elicit a transformed phenotype in these cells.

Transforming growth factor-beta induces cytoskeleton and extracellular matrix modifications in FRTL-5 thyroid epithelial cells.

The action of transforming growth factor-beta (TGF-beta) on the morphology, cytoskeleton and extracellular matrix was investigated in FRTL-5 thyroid epithelial cells. After treatment with TGF-beta, FRTL-5 cells became flat and developed straight and thick bundles of actin microfilaments. This effect of TGF-beta was observed even in the presence of thyrotropin, which has a strong microfilament disrupting action. TGF-beta also influenced some aspects of the extracellular matrix organization. Immunofluorescence staining of FRTL-5 cells revealed both the appearance of a fibrillar array of fibronectin in association with the basal plasma membrane and a change in the morphology of basally located laminin patches. TGF-beta induced the formation of adhesion structures at the ventral portion of the cell membrane. Vinculin was focally concentrated at the end of stress fibers in areas corresponding to focal adhesions as revealed by interference reflection microscopy (IRM). The ability to modulate cytoskeleton organization and extracellular matrix protein distribution might mediate some of the reported TGF-beta effects on the expression of specific functional properties in thyroid cells.

Thyrotropin receptor gene expression in oncogene-transfected rat thyroid cells: correlation between transformation, loss of thyrotropin-dependent growth, and loss of thyrotropin receptor gene expression.

Rat FRTL-5 and PC-Cl-3 thyroid cells are continuously cultured, clonal lines which require thyrotropin to grow and function. Both can be efficiently transformed when infected with RNA or DNA viruses carrying oncogenes or when directly transfected with activated oncogenes. Transformation, assayed by the appearance of cell growth in agar and by tumorigenicity in syngeneic rats or nude mice, is associated with the loss of thyrotropin-dependent cell division and thyrotropin-regulated functions such as thyroglobulin synthesis. In 16 clones of FRTL-5 or PC-Cl-3 cells transformed with different oncogenes, we show that loss of thyrotropin-dependent growth and function correlates with the loss of thyrotropin receptor gene expression, measured with a rat thyrotropin receptor cDNA probe.

Thyrotropin stimulates transcription from the ferritin heavy chain promoter.

Thyrotropin (TSH) is the primary hormone regulating the activity of the thyroid gland. We have recently shown that TSH stimulates H-ferritin mRNA levels in rat thyroid. Ferritin plays a key role in determining the intracellular fate of iron. The induction of ferritin synthesis by iron in liver is regulated both at transcriptional and translational levels. Here we present evidence that the mechanisms by which TSH regulates the mRNA levels are mediated by a diffusible product acting in trans on its own promoter. In fact, the H-ferritin promoter mediates increased CAT activity in response to hormone induction. Our results identify transcription as an important regulatory step of TSH action. They suggest that TSH induces expression of the ferritin gene, and that continuous protein synthesis is required to maintain basal ferritin gene expression in the absence of hormone.

Dual effect of transforming growth factor beta on rat thyroid cells: inhibition of thyrotropin-induced proliferation and reduction of thyroid-specific differentiation markers.

Transforming growth factor beta (TGF beta) is now known to have a number of effects other than inducing phenotypic transformation of fibroblastic cells: TGF beta controls proliferation, differentiation, and other functions in many cell types. In this paper we have analyzed the action of TGF beta 1 on a system of differentiated epithelial rat thyroid cells in culture (FRTL5), which depend on the addition of thyrotropin for their growth. TGF beta 1 is able to inhibit the growth of thyroid cells by reducing cell response to thyrotropin. Moreover, in analogy to the effect produced upon other differentiated cells in culture, such as myoblasts and adipocytes, TGF beta 1 modulates the expression of FRTL5-specific thyroid markers, reducing thyroglobulin biosynthesis and the ability to concentrate the iodide.

Adenylate cyclase activity of v-ras-k transformed rat epithelial thyroid cells.

The regulation of adenylate cyclase has been analyzed in normal rat thyroid cells as well as in the same cells transformed by the v-ras-k oncogene. In both cell types the adenylate cyclase complex consists of the two GTP-binding proteins, Gi and Gs, as demonstrated by the specific ADP-ribosylation induced by pertussis and cholera toxin, respectively. The response of adenylate cyclase of the transformed cells to forskolin, pertussis toxin and cholera toxin is attenuated with respect to the control cell line. The thyrotropic hormone (TSH), that acts on normal thyroid cells in culture as a growth factor by stimulating the adenylate cyclase activity, is not able to induce DNA synthesis nor does it stimulate adenylate cyclase in v-ras-k transformed cells.

Forskolin and a tumor promoter are able to induce c-fos and c-myc expression in normal, but not in a v-ras-transformed rat thyroid cell line.

The rat thyroid cell line (FRTL5) is dependent on thyrotropic hormone (TSH) for its growth. c-fos and c-myc oncogenes expression was measured in these cells after addition of their specific growth factor TSH and after treatment with either forskolin, an activator of adenylate cyclase or with a tumor promoter, TPA. Transient expression of oncogenes coding for nuclear products and a slight increase in ras-h oncogene expression were observed in normal rat thyroid cells after all treatments. In contrast, in v-ras-transformed rat thyroid cells, which express very high levels of p21, treatment with either TSH, forskolin or TPA does not induce c-fos gene expression, while c-myc expression was constitutive. Normal unstimulated cells show no c-myc expression.

Induction of the c-fos oncogene by thyrotropic hormone in rat thyroid cells in culture.

Rat thyroid cells in culture, rendered quiescent by hormone deprivation, can be stimulated to undergo DNA synthesis in the absence of serum by the addition of purified thyrotropin. The primary effect in response to thyrotropin action in thyroid cells is the induction of the c-fos oncogene, followed by c-myc expression. This suggests that thyrotropin acts as a competence growth factor.