A novel NDUFV1 gene mutation in complex I deficiency in consanguineous siblings with brainstem lesions and Leigh syndrome.

Although deficiency of complex I of the mitochondrial respiratory chain is a frequent cause of encephalopathy in children, only a few mutations have been reported in each of its subunits. In the absence of families large enough for conclusive segregation analysis and of robust functional testing, it is difficult to unequivocally show the causality of the observed mutations and to delineate genotype-phenotype correlations, making additional observations necessary. We observed two consanguineous siblings with an early-onset encephalopathy, medulla, brainstem and mesencephalon lesions on brain magnetic resonance imaging and death before 8 months of age, caused by a complex I deficiency. We used a homozygosity mapping approach and identified a missense mutation in the NDUFV1 gene. The mutation, p.Arg386His, affects a highly conserved residue, contiguous to a cysteine residue known to coordinate an Fe ion. This observation adds to our understanding of complex I deficiency disease. It validates the important role of Arg386 and therefore supports the current molecular model of iron-sulfur clusters in NDUFV1.

Cross signaling, cell specificity, and physiology.

The literature on intracellular signal transduction presents a confusing picture: every regulatory factor appears to be regulated by all signal transduction cascades and to regulate all cell processes. This contrasts with the known exquisite specificity of action of extracellular signals in different cell types in vivo. The confusion of the in vitro literature is shown to arise from several causes: the inevitable artifacts inherent in reductionism, the arguments used to establish causal effect relationships, the use of less than adequate models (cell lines, transfections, acellular systems, etc.), and the implicit assumption that networks of regulations are universal whereas they are in fact cell and stage specific. Cell specificity results from the existence in any cell type of a unique set of proteins and their isoforms at each level of signal transduction cascades, from the space structure of their components, from their combinatorial logic at each level, from the presence of modulators of signal transduction proteins and of modulators of modulators, from the time structure of extracellular signals and of their transduction, and from quantitative differences of expression of similar sets of factors.

The visual display of regulatory information and networks.

Cell regulation and signal transduction are becoming increasingly complex, with reports of new cross-signalling, feedback, and feedforward regulations between pathways and between the multiple isozymes discovered at each step of these pathways. However, this information, which requires pages of text for its description, can be summarized in very simple schemes, although there is no consensus on the drawing of such schemes. This article presents a simple set of rules that allows a lot of information to be inserted in easily understandable displays.

Transforming growth factor beta(1) selectively inhibits the cyclic AMP-dependent proliferation of primary thyroid epithelial cells by preventing the association of cyclin D3-cdk4 with nuclear p27(kip1).

Dog thyroid epithelial cells in primary culture constitute a physiologically relevant model of positive control of DNA synthesis initiation and G0-S prereplicative phase progression by cAMP as a second messenger for thyrotropin (thyroid-stimulating hormone [TSH]). As previously shown in this system, the cAMP-dependent mitogenic pathway differs from growth factor cascades as it stimulates the accumulation of p27(kip1) but not cyclins D. Nevertheless, TSH induces the nuclear translocations and assembly of cyclin D3 and cdk4, which are essential in cAMP-dependent mitogenesis. Here we demonstrate that transforming growth factor beta(1) (TGFbeta(1)) selectively inhibits the cAMP-dependent cell cycle in mid-G1 and various cell cycle regulatory events, but it weakly affects the stimulation of DNA synthesis by epidermal growth factor (EGF), hepatocyte growth factor, serum, and phorbol esters. EGF+serum and TSH did not interfere importantly with TGFbeta receptor signaling, because they did not affect the TGFbeta-induced nuclear translocation of Smad 2 and 3. TGFbeta inhibited the phosphorylation of Rb, p107, and p130 induced by TSH, but it weakly affected the phosphorylation state of Rb-related proteins in EGF+serum-treated cells. TGFbeta did not inhibit c-myc expression. In TSH-stimulated cells, TGFbeta did not affect the expression of cyclin D3, cdk4, and p27(kip1), nor the induced formation of cyclin D3-cdk4 complexes, but it prevented the TSH-induced relocalization of p27(kip1) from cdk2 to cyclin D3-cdk4. It prevented the nuclear translocations of cdk4 and cyclin D3 without altering the assembly of cyclin D3-cdk4 complexes probably formed in the cytoplasm, where they were prevented from sequestering nuclear p27(kip1) away from cdk2. This study dissociates the assembly of cyclin D3-cdk4 complexes from their nuclear localization and association with p27(kip1). It provides a new mechanism of regulation of proliferation by TGFbeta, which points out the subcellular location of cyclin D-cdk4 complexes as a crucial factor integrating mitogenic and antimitogenic regulations in an epithelial cell in primary culture.

Characterization of autonomous thyroid adenoma: metabolism, gene expression, and pathology.

Fifty-one in vivo characterized autonomous single adenomas have been studied for functional parameters in vitro, for gene and protein expression and for pathology, and have been systematically compared to the corresponding extratumoral quiescent tissue. The adenomas were characterized by a high level of iodide trapping that corresponds to a high level of Na+ /iodide symporter gene expression, a high thyroperoxidase mRNA and protein content, and a low H2O2 generation. This explains the iodide metabolism characteristics demonstrated before, ie, the main cause of the "hot" character of the adenomas is their increased iodide transport. The adenomas spontaneously secreted higher amounts of thyroid hormone than the quiescent tissue and in agreement with previous in vivo data, this secretion could be further enhanced by thyrotropin (TSH). Inositol uptake was also increased but there was no spontaneous increase of the generation of inositol phosphates and this metabolism could be further activated by TSH. These positive responses to TSH are in agreement with the properties of TSH-stimulated thyroid cells in vitro and in vivo. They are compatible with the characteristics of mutated TSH receptors whose constitutive activation accounts for the majority of autonomous thyroid adenomas in Europe. The number of cycling cells, as evaluated by MIB-1 immunolabeling was low but increased in comparison with the corresponding quiescent tissue or normal tissue. The cycling cells are observed mainly at the periphery; there was very little apoptosis. Both findings account for the slow growth of these established adenomas. On the other hand, by thyroperoxidase immunohistochemistry, the whole lesion appeared hyperfunctional, which demonstrates a dissociation of mitogenic and functional stimulations. Thyroglobulin, TSH receptor, and E-cadherin mRNA accumulations were not modified in a consistent way, which confirms the near-constitutive expression of the corresponding genes in normal differentiated tissue. On the contrary, early immediate genes expressions (c-myc, NGF1B, egr 1, genes of the fos and jun families) were decreased. This may be explained by the proliferative heterogeneity of the lesion and the previously described short, biphasic expression of these genes when induced by mitogenic agents. All the characteristics of the autonomous adenomas can therefore be explained by the effect of the known activating mutations of genes coding for proteins of the TSH cyclic adenosine monophosphate (cAMP) cascade, all cells being functionally activated while only those at the periphery multiply. The reason of this heterogeneity is unknown.

Immediate early gene expression in dog thyrocytes in response to growth, proliferation, and differentiation stimuli.

In dog thyroid cells, insulin or IGF-1 induces cell growth and is required for the mitogenic action of TSH through cyclic AMP, of EGF, and of phorbol esters. HGF per se stimulates cell proliferation and is thus the only full mitogenic agent. TSH and cAMP enhance, whereas EGF phorbol esters and HGF repress differentiation expression. In this study, we have investigated for each factor and regulatory cascade of the intermediate step of immediate early gene induction, that is, c-myc, c-jun, jun D, jun B, c-fos, fos B, fra-1, fra-2, and egr1; fra-1 and fra-2 expressions were very low. TSH or forskolin increased the levels of c-myc, jun B, jun D, c-fos, and fos B while decreasing those of c-jun and egr1. Phorbol myristate ester stimulated the expression of all the genes. EGF and HGF stimulated the expression of all the genes except jun D and for EGF fos B. All these effects were obtained in the presence and in the absence of insulin, which shows that insulin is not necessary for the effects of the mitogens on immediate early gene expression. The definition of the repertoire of early immediate genes inductible by the various growth cascades provides a framework for the analysis of gene expression in tumors. (1) Insulin was able to induce all the protooncogenes investigated except fos B. This suggests that fos B could be the factor missing for insulin to induce mitogenesis. (2) No characteristic pattern of immediate early gene expression has been observed for insulin, which induces cell hypertrophy and is permissive for the action of the other growth factors. These effects are therefore not accounted for by a specific immediate early gene expression. On the other hand, insulin clearly enhances the effects of TSH, phorbol ester, and EGF on c-myc, junB, and c-fos expression. This suggests that the effect of insulin on mitogenesis might result from quantitative differences in the transcription complexes formed. (3) c-myc, c-fos, and jun B mRNA induction by all stimulating agents, whether inducing cell hypertrophy, or growth and dedifferentiation, or growth and differentiation, suggests that, although these expressions are not sufficient, they may be necessary for the various growth responses of thyroid cells. (4) The inhibition of c-jun and egr1 mRNA expression, and the marked induction of jun D mRNA appear to be specific features of the TSH cAMP pathway. They might be related to its differentiating action. (5) fos B, which is induced by TSH, forskolin, phorbol ester, and HGF but not by insulin, could be involved in the mitogenic action of the former factors.

Use of two-hybrid methodology for identifying proteins of interest in endocrinology.

During the last 10 years, much progress has been made in understanding signal transduction. However, the function of many newly identified proteins remains unknown. The protein/protein interactions have emerged as a major biochemical mechanism of signal transduction. They are of major interest to elucidate the role of a protein in one or another cellular process. The two-hybrid system is especially well designed for such investigation. Here we show that the contribution of this technique already is and will be essential in dissecting the molecular mechanism of transduction pathways in many cell types.

Identification and characterization of mRNAs differentially expressed in thyroid cells stimulated by a mitogenic treatment.

The aim of our work is to identify new genes and proteins involved in the control of the proliferation of thyroid cells as putative protooncogenes and antioncogenes. Several strategies are discussed. A first study has allowed to identify three new genes. Further search will use the differential display and gene arrays methodology. The role of the identified proteins coded by the genes is studied in vitro by the search of partner proteins by the double hybrid method and in vivo by mice gene knockout technology.

IGF-1 or insulin, and the TSH cyclic AMP cascade separately control dog and human thyroid cell growth and DNA synthesis, and complement each other in inducing mitogenesis.

The regular doubling of cell mass, and therefore of cell protein content, is required for repetitive cell divisions. Preliminary observations have shown that in dog thyrocytes insulin induces protein accumulation but not DNA synthesis, while TSH does not increase protein accumulation but triggers DNA synthesis in the presence of insulin. We show here that EGF and phorbol myristate ester complement insulin action in the same way. HGF is the only factor activating both protein accumulation and DNA synthesis. The effects of insulin on protein accumulation and in permitting the TSH effect are reproduced by IGF-1 and are mediated, at least in part by the IGF-1 receptor. The concentration effect curves are similar for both effects. Similar results are obtained in human thyrocytes. They reflect true cell growth, as shown by increases in RNA content and cell size. Carbachol and fetal calf serum also stimulate protein synthesis and accumulation without triggering DNA synthesis, but they are not permissive for the mitogenic effects of TSH or of the general adenylate cyclase activator, forskolin. Moreover the mitogenic effect of TSH greatly decreased in cells deprived of insulin for 2 days although these cells remain hypertrophic. Hypertrophy may therefore be necessary for cell division, but it is not sufficient to permit it. Three different mechanisms can therefore be distinguished in the mitogenic action of TSH: (1) the increase of cell mass (hypertrophy) induced by insulin or IGF-1; (2) the permissive effect of insulin or IGF-1 on the mitogenic effect of TSH which may involve both the increase of cell mass and the induction of specific proteins such as cyclin D3 and (3) the mitogenic effect of the TSH cyclic AMP cascade proper.

Induction of nerve growth factor-induced gene-B (NGFI-B) as an early event in the cyclic adenosine monophosphate response of dog thyrocytes in primary culture.

We investigated the induction of nerve growth factor-induced gene-B (NGFI-B) in dog thyrocytes in primary culture stimulated by different agents. The dog NGFI-B complementary DNA (cDNA) was cloned from a cDNA library of dog thyrocytes and used to study, by Northern blotting, the level of NGFI-B messenger RNA (mRNA) in those cells. We have shown that TSH and forskolin, which both induce proliferation and differentiation of the thyroid cells by activation of the protein kinase A pathway, lead to a strong and transient expression of two NGFI-B mRNA species, which differ in the length of the poly(A) tail. In contrast, 12-O-tetradecanoyl-13-phorbol-acetate (TPA) and epidermal growth factor, which induce proliferation and dedifferentiation of those cells by activation of the protein kinase C and the protein tyrosine kinase cascade, respectively, lead to a weaker expression of NGFI-B mRNA. In parallel, we studied the transactivation capacity of NGFI-B in the same cell system by transient transfection of a chloramphenicol acetyl transferase reporter construction containing a NGFI-B-dependent synthetic promoter. The highest transactivation was observed after forskolin stimulation, whereas transactivation after TPA stimulation was weak and no significant transactivation was observed after epidermal growth factor stimulation. Taken together, these results show that NGFI-B is an immediate early gene product that is mainly induced by the cAMP-dependent pathway in dog thyrocytes. Moreover they suggest that NGFI-B expression could be one of the early transcriptional changes induced specifically by this cascade and leading to differentiation and/or proliferation of these cells.

Thyrotropin via cyclic AMP induces insulin receptor expression and insulin Co-stimulation of growth and amplifies insulin and insulin-like growth factor signaling pathways in dog thyroid epithelial cells.

Despite the similarity of their receptors and signal transduction pathways, insulin is regarded as a regulator of glucose, protein, and lipid metabolism, whereas insulin-like growth factors (IGF-I and IGF-II) mainly act as mitogenic hormones. In the dog thyroid primary culture model, the triggering of DNA synthesis by thyrotropin (TSH) through cAMP, or by cAMP-independent factors including epidermal growth factor, hepatocyte growth factor and phorbol esters, requires insulin or IGFs as comitogenic factors. In the present study, in TSH-treated cells, IGF-I receptors and insulin receptors were paradoxically equivalent in their capacity to elicit the comitogenic pathway, which, however, was mediated only by IGF-I receptors in dog thyroid cells stimulated by cAMP-independent mitogens. Moreover, prior cell exposure to TSH or forskolin increased their responsiveness to insulin, IGF-I, and IGF-II, as seen on DNA synthesis and activation of a common insulin/IGF signaling pathway. To understand these observations, binding characteristics and expression of insulin and IGF-I receptors were examined. To analyze IGF-I receptor characteristics, the unexpected interference of a huge presence of IGF-binding proteins at the cell membrane was avoided using labeled Long R3 IGF-I instead of IGF-I. Strikingly, TSH, through cAMP, time-dependently induced insulin binding and insulin receptor mRNA and protein accumulation without any effect on IGF-I receptors. These findings constitute a first example of an induction of insulin receptor gene expression by a cAMP-mediated hormone. In dog thyroid cells, this allows low physiological insulin concentrations to act as a comitogenic factor and might explain in part the enhanced responsiveness to IGFs in response to TSH. This raises the possibility that TSH-insulin interactions may play a role in the regulation of thyroid growth and function in vivo.

c-Myc expression is controlled by the mitogenic cAMP-cascade in thyrocytes.

In dog thyroid epithelial cells in primary culture, thyrotropin (TSH), acting through cAMP, induces proliferation and differentiation expression, whereas epidermal growth factor (EGF) and phorbol esters induce proliferation and dedifferentiation. In these cells, we have detailed the regulation by cAMP of the c-myc protooncogene mRNA and protein. The cAMP signaling pathway induces a biphasic increase of c-myc mRNA and protein. c-Myc protein accumulation follows the abundance and kinetics of its mRNA expression. Using in vitro elongation of nascent transcripts to measure transcription and actinomycin D (AcD) chase experiments to study mRNA stability, we have shown that in the first phase cAMP releases a transcriptional elongation block. No modification of transcriptional initiation was observed. After 30 min of treatment with TSH, c-myc mRNA was also stabilized. During the second phase, cAMP stabilization of the mRNA disappears and transcription is again shut off. Thus, in a tissue in which it stimulates proliferation and specific gene expression, cAMP regulates biphasically c-myc expression by mechanisms operating at the transcriptional and posttranscriptional levels.

Jun B expression is regulated differently by three mitogenic pathways in thyrocytes.

In dog thyrocytes in primary culture, thyrotropin (TSH), acting through cyclic AMP, induces proliferation and differentiation expression, while tetradecanoylphorbol acetate (TPA) or epidermal growth factor (EGF) induces proliferation and dedifferentiation. In this work, we have investigated the regulation of mRNA expression of the protooncogene jun B in these cells. TSH stimulated jun B expression very transiently with biphasic kinetics similar to those obtained for c-myc mRNA accumulation. Forskolin reproduced these effects, suggesting that they are, as other effects of TSH in this system, mediated by cyclic AMP. As shown by nuclear run-on experiments, jun B is regulated by the cAMP pathway at the transcriptional level. As in other cell types, EGF or TPA caused a more sustained increase in mRNA levels. In thyroid slices, in which DNA synthesis appears to be induced by the wounding process, jun B is also induced, suggesting a correlation with the proliferative status of the cell. Interestingly, two jun B mRNAs of 2.1 and 2.3 kb were induced by all the mitogenic pathways. The kinetics of their accumulation were different; i.e., TPA induced the smaller transcript with some delay after the longer one and cycloheximide induced the progressive shortening of the first appearing heavier mRNA. The 2.3-kb messenger has a longer poly(A) tail, and kinetics in the presence of actinomycin D suggested it could represent a precursor form of the 2.1-kb messenger. It is suggested that the specific kinetics of cyclic-AMP-induced accumulation of jun B mRNA could be related to the dual stimulation of differentiation and proliferation by TSH in dog thyrocytes.