Involvement of mTOR and Regulation by AMPK in Early Iodine Deficiency-Induced Thyroid Microvascular Activation.

Iodine deficiency (ID) induces TSH-independent microvascular activation in the thyroid via the reactive oxygen species/nitric oxide-hypoxia-inducible factor-1α/vascular endothelial growth factor (VEGF) pathway. We hypothesized the additional involvement of mammalian target of rapamycin (mTOR) as a positive regulator of this pathway and AMP-activated protein kinase (AMPK) as a negative feedback regulator to explain the transient nature of ID-induced microvascular changes under nonmalignant conditions. mTOR and AMPK involvement was investigated using an in vitro model (human thyrocytes in primary cultures) and 2 murine models of goitrogenesis (normal NMRI and RET-PTC mice [a papillary thyroid cancer model]). In NMRI mice, ID had no effect on the phosphorylation of ribosomal S6 kinase (p70S6K), a downstream target of mTOR. However, rapamycin inhibited ID-induced thyroid blood flow and VEGF protein expression. In the RET-PTC model, ID strongly increased the phosphorylation of p70S6K, whereas rapamycin completely inhibited the ID-induced increase in p70S6K phosphorylation, thyroid blood flow, and VEGF-A expression. In vitro, although ID increased p70S6K phosphorylation, the ID-stimulated hypoxia-inducible factor/VEGF pathway was inhibited by rapamycin. Activation of AMPK by metformin inhibited ID effects both in vivo and in vitro. In AMPK-α1 knockout mice, the ID-induced increase in thyroid blood flow and VEGF-A protein expression persisted throughout the treatment, whereas both parameters returned to control values in wild-type mice after 4 days of ID. In conclusion, mTOR is required for early ID-induced thyroid microvascular activation. AMPK negatively regulates this pathway, which may account for the transient nature of ID-induced TSH-independent vascular effects under benign conditions.

The Ca(2+) /calmodulin-dependent kinase kinase ß-AMP-activated protein kinase-α1 pathway regulates phosphorylation of cytoskeletal targets in thrombin-stimulated human platelets.

BACKGROUND: Platelet activation requires sweeping morphologic changes, supported by contraction and remodeling of the platelet actin cytoskeleton. In various other cell types, AMP-activated protein kinase (AMPK) controls the phosphorylation state of cytoskeletal targets. OBJECTIVE: To determine whether AMPK is activated during platelet aggregation and contributes to the control of cytoskeletal targets. RESULTS: We found that AMPK-α1 was mainly activated by thrombin, and not by other platelet agonists, in purified human platelets. Thrombin activated AMPK-α1 ex vivo via a Ca(2+) /calmodulin-dependent kinase kinase ß (CaMKKß)-dependent pathway. Pharmacologic inhibition of CaMKKß blocked thrombin-induced platelet aggregation and counteracted thrombin-induced phosphorylation of several cytoskeletal proteins, namely, regulatory myosin light chains (MLCs), cofilin, and vasodilator-stimulated phosphoprotein (VASP), three key elements involved in actin cytoskeletal contraction and polymerization. Platelets isolated from mice lacking AMPK-α1 showed reduced aggregation in response to thrombin, and this was associated with defects in MLC, cofilin and VASP phosphorylation and actin polymerization. More importantly, we show, for the first time, that the AMPK pathway is activated in platelets of patients undergoing major cardiac surgery, in a heparin-sensitive manner. CONCLUSION: AMPK-α1 is activated by thrombin in human platelets. It controls the phosphorylation of key cytoskeletal targets and actin cytoskeletal remodeling during platelet aggregation.

Antagonistic effects of leucine and glutamine on the mTOR pathway in myogenic C2C12 cells.

This study compared the effects of leucine and glutamine on the mTOR pathway, on protein synthesis and on muscle-specific gene expression in myogenic C(2)C(12) cells. Leucine increased the phosphorylation state of mTOR, on both Ser2448 and Ser2481, and its downstream effectors, p70(S6k), S6 and 4E-BP1. By contrast, glutamine decreased the phosphorylation state of mTOR on Ser2448, p70(S6k) and 4E-BP1, but did not modify the phosphorylation state of mTOR on Ser2481 and S6. Whilst the phosphorylation state of the mTOR pathway is usually related to protein synthesis, the incorporation of labelled methionine/cysteine was only transiently modified by leucine and was unaltered by glutamine. However, these two amino acids affected the mRNA levels of desmin, myogenin and myosin heavy chain in a time-dependant manner. In conclusion, leucine and glutamine have opposite effects on the mTOR pathway. Moreover, they induce modification of muscle-specific gene expression, unrelated to their effects on the mTOR/p70(S6k) pathway.

New targets of AMP-activated protein kinase.

The discovery of the AMP-activated protein kinase (AMPK) more than a decade ago has shed much light on the cellular response to stresses characterized by a fall in the concentration of ATP and an increase in the AMP/ATP ratio. All conditions known to increase this ratio activate AMPK, whose major role is to act as an emergency signal to conserve ATP. It does so by inhibiting anabolic processes and by activating pathways producing ATP. In recent years, our laboratory has discovered new targets of AMPK. The purpose of this short review is to summarize our contribution to this field.

MCF-7 mammary tumour cells express the myeloid cell differentiation controlling factor, serine protease 3/myeloblastin.

Our previous data indicated that HSP27 plays a role in MCF-7 cell differentiation similar to that it has in HL-60 cells. In the latter case, this involves a control of its levels by proteinase 3/myeloblastin (PR3/Mbn), a serine proteinase hitherto considered specific of the myeloid lineage. Having observed that the treatment of MCF-7 cells with the serine protease inhibitor N-tosyl-l-phenylalanine-chloromethyl ketone (TPCK) increased their content in HSP27 and induced them to acquire a secretory phenotype, we undertook this work to test the assumption that an enzyme similar or identical to PR3/Mbn might be expressed in this cell line. The data show that MCF-7 cells exhibited specific immunopositivity for a monoclonal antibody against PR3/Mbn. Western blot analysis of immunoprecipitates from MCF-7 cell extracts, obtained and checked with PR3/Mbn monoclonal antibodies, confirmed the presence of the 35 kDa glycosylated and 29 kDa mature forms of the protein. Finally, Northern blot analysis confirmed the expression of the corresponding mRNA. Together with our data with TPCK, this substantiates our hypothesis that, as in HL-60 cells, regulation of MCF-7 cells differentiation might involve a postranslation control on HSP27 levels by a serine protease.

Effect of the serine protease inhibitor N-tosyl-l-phenylalanine-chloromethyl ketone (TPCK) on MCF-7 mammary tumour cells growth and differentiation.

Previous studies from this and other laboratories indicated that the oestrogen-regulated heat shock protein HSP27 is involved in the control of MCF-7 cells growth and differentiation, as it also appears to be in other cell types, including osteoblasts and HL-60 cells. In the latter instance, induction of differentiation is associated with the downregulation of myeloblastin, a serine protease now identified as proteinase 3 (hence its designation as PR3/Mbn), mirrored by an increase in the cellular content of the small heat shock protein HSP27, a substrate to this enzyme. Besides, antisense inhibition of PR3/Mbn production sufficed for inducing HL-60 cells monocytic differentiation. This prompted us to examine the hypothesis that a post-translational control on HSP27 levels (and by this on differentiation) by a serine protease might also be operating in human mammary tumour cells. As part of our attempt to evaluate this hypothesis, the present work consisted of testing the effects of a treatment of MCF-7 cells with the serine protease inhibitor N-tosyl-L-phenylalanine-chloromethyl ketone (TPCK). Our data show that this resulted in a four-fold increase in HSP27 content, associated with a 2.5-fold decrease in growth rate, the formation of cytoplasmic vesicles and increased secretion of 52 kDa peptides, identified by Western immunoblot as the isoforms of the oestrogen-regulated protein, cathepsin D. TPCK only affected growth in MDAMB-231 cells (in which HSP27 levels are very low and remained below MCF-7 cells basal levels after treatment) and failed to affect L929 cells, in which the hsp27 gene is silent. This provides circumstantial support for the assumption that effects of TPCK on the MCF-7 cells phenotype are linked to the associated increase in HSP27 content. Our recent demonstration that MCF-7 cells do in fact express PR3/Mbn fits with our concept and opens the way to test it directly, using antisense strategy.

Anti-sense inhibition of small-heat-shock-protein (HSP27) expression in MCF-7 mammary-carcinoma cells induces their spontaneous acquisition of a secretory phenotype.

This work was aimed at testing the hypothesis (hitherto supported only by indirect evidence) that, besides contributing to resistance to stress, the small heat-shock-protein HSP27 might be involved in the control of growth and differentiation in mammary-tumour cells, where it is known to be oestrogen-regulated. Therefore, MCF-7 cells were transfected with a modulatable human hsp27 anti-sense cDNA. Clones of transfectants (designated alphahsp27) were selected which, upon expression of the anti-sense, exhibited a decline in HSP27 accumulation, associated with a decrease in resistance to heat shock and in proliferation rate, the degree of the latter reflecting their respective reduction in HSP27 content. The effects of anti-sense inhibition of HSP27 production were similar to those exerted on parental cells by phorbol myristate (TPA). Both resulted in growth inhibition, accumulation of lipid droplets in the cytoplasm, formation of secretory microvesicles with internal microvilli and increased release of several proteins, including the isoforms of a 52-kDa protein, which we identified as the oestrogen-regulated protein cathepsin D, all this without noticeable change in actin organization. These data constitute the first direct support for the hypothesis that, at least in some cell types, HSP27 might play a modulatory role in cell differentiation and (perhaps by this) in proliferation. While allowing dissociation of this role from the known action of HSP27 on actin polymerization, they suggest similar modulation of the function of some protein(s) implicated in the acquisition of the secretory phenotype by MCF-7 cells, with HSP27 also exerting an inhibitory action that can be alleviated either by its phosphorylation (as occurs with TPA) or by inhibition of its production.

Expression of HSP27 results in increased sensitivity to tumor necrosis factor, etoposide, and H2O2 in an oxidative stress-resistant cell line.

The role of HSP27 in cell growth and resistance to stress was investigated using murine fibrosarcoma L929 cells (normally devoid of constitutively expressed small HSPs) and human osteoblast-like SaOS-2 cells stably transfected with a human hsp27 expression vector. Our data showed that our L929 cells were more resistant to oxidative stress than generally observed for this line. Production of HSP27 in these cells led to a marked decrease in growth rate associated with a series of phenotypical changes, including cell spreading, cellular and nuclear hypertrophy, development of an irregular outline, and a tremendous accumulation of actin stress fibers. By contrast, none of these changes was observable in SaOS-2/hsp27 transfectants overexpressing the protein product. Together, these observations are consistent with a cause-to-effect cascade relationship between increased (or induced) HSP27 expression, changes in cytoskeletal organization, and decreased growth. On the other hand, whereas the transfection of the hsp27 gene increased the cell resistance to heat in both cell lines, only in SaOS-2 cells was this associated with protection to the cytotoxic action of tumor necrosis factor-alpha (TNF-alpha) and etoposide. Unexpectedly, L929/hsp27 transfectants exhibited an increased sensitivity to both agents and also to H2O2. These data thus imply that different mechanisms are involved in the cell resistance to heat shock and to the cytotoxic action of TNF-alpha, etoposide, and H2O2. They also plead against the simple view that overexpression of a phosphorylatable HSP27 would necessarily be beneficial in terms of increased cell resistance to any type of stress. Our data further indicate that the role of HSP27 in cellular resistance to stress and in cell proliferation involves different targets and that the ultimate result of its interference with these processes depends on the intracellular context in which the protein is expressed.

Changes in the phosphorylation status of the 27 kDa heat shock protein (HSP27) associated with the modulation of growth and/or differentiation in MCF-7 cells.

We have used human mammary cells of the MCF-7 strain, which constitutively express high levels of the small heat shock protein HSP27 and we have compared the changes in the phosphorylation status of this protein together with changes in cell growth and/or morphology induced by the action of one of the following agents: (1) TPA (12-O-tetradecanoylphorbol-13-acetate), known as a differentiation inducer in MCF-7 cells; (2) OH-TAM (hydroxytamoxifen), which exerts a cytostatic and cytotoxic action; or (3) TNF alpha (tumour necrosis factor), which induces apoptotic cell death in this cell line. Our data show that TPA and TNF stimulate an immediate and massive phosphorylation of HSP27, whereas OH-TAM affect the phosphorylation status of the protein only after a 3 day delay. In the case of TPA, high levels of HSP27 phosphorylation were maintained for at least 4 days, along with growth inhibition and acquisition by the cells of a secretory phenotype. TPA and OH-TAM exerted similar immediated effects on cell growth, despite the different time course of their action on HSP27 phosphorylation. This excludes the possibility that the latter is a necessary consequence of, or an absolute requisite to, growth inhibition. With OH-TAM and TNF the increase in HSP27 phosphorylation was concomitant with the appearance of apoptosis, not observed with TPA. This indicates that increased phosphorylation of HSP27 is not specifically associated with the triggering or the execution of apoptosis in these cells. Altogether, our data support the concept that phosphorylated HSP27 is involved (and might then be rate limiting in some instances) in the execution of vital cell programmes (including resistance to stress, proliferation and differentiation), as well as in that of cell death. This is consistent with its role in actin polymerization and its position downstream of the p38/RK-type MAPkinase, itself a point of convergence for diverse signal transduction pathways.

Antisense inhibition of the 27 kDa heat shock protein production affects growth rate and cytoskeletal organization in MCF-7 cells.

MCF-7 cells were co-transfected with the human HSP27 antisense cDNA and the neomycin resistance gene, included in the constitutive expression vector pSVL, and the phenotypical changes associated with decreased expression of the HSP27 protein were analysed. Three out of 10 neomycin-resistant clones obtained proliferated normally and showed a normal HSP27 content (Western blot). The seven other clones (designated as alpha HSP27 clones) were characterized by a dramatic growth inhibition associated with alterations in cellular morphology. Cells became progressively hypertrophied, exhibited lamellar protrusions and tended to lose contact with each other. They also acquired characteristics of secretory cells, namely the presence of numerous refractile granules and secretory canaliculi. Among the alpha HSP27 clones, two were immunocytochemically analysed for HSP27 content. Both clones were immunonegative for HSP27, contrary to parental cells and neo-transfectants. Actin immunostaining in one of these HSP27 negative clones revealed that microfilament organization changed from diffuse to punctate distribution. Our data support the current concept of a role for HSP27 in cell growth and differentiation and further suggests that this might occur through a control on actin polymerization-depolymerization.

The role of social support on health throughout the lifecycle.

The role of social supports throughout the lifecycle has dynamic implications on health and psychosocial wellness. Establishment of a stable supportive family early in life seems to be a consistent support drawn upon throughout the lifecycle. Other social support predictors which may affect health include high-quality supports from friends, total network size, age, gender, marital status, income, and threatening life events. Implications of social supports throughout the lifecycle should encourage health educators of all types to promote quality social supports within the family, schools, and community.