No abstract

REK7 is an Eph-related tyrosine kinase receptor expressed exclusively in the nervous system, predominantly in hippocampus and cortex. A soluble REK7-IgG fusion protein, produced to analyze the biological role of REK7, prevents axon bundling in cocultures of cortical neurons with astrocytes, a model of late stage nervous system development and differentiation. Using REK7-IgG as an affinity reagent, we purified and cloned a novel REK7 ligand called AL-1, a GPI-linked protein homologous to other members of an emerging ligand family. Membrane attachment of AL-1 appears necessary for receptor activation, since REK7 on cortical neurons is efficiently activated by transfected cells expressing GPI-linked AL-1, but not by soluble AL-1. Consistent with this, soluble AL-1 blocks axon bundling. Our findings, together with the observation that both molecules are expressed in the brain, suggest a role in the formation of neuronal pathways, a crucial feature of nervous system development and regeneration.

The stilbene resveratrol (RV) initiates p53-dependent apoptosis via plasma membrane integrin aVb3 in human cancer cells. A thyroid hormone (L-thyroxine, T 4 ) membrane receptor also exists on aVb3. Stilbene and T 4 signals are both transduced by extracellular-regulated kinases 1 and 2 (ERK1/2); however, T 4 promotes cell proliferation in cancer cells, whereas RV is proapoptotic. Thyroid hormone has been shown to interfere with RV-induced apoptosis. However, the mechanisms involved are not fully understood. In this study, we examined the mechanism whereby T 4 inhibits RV-induced apoptosis in glioma cells. RV activated conventional protein kinase C and ERK1/2 and caused nuclear localization of cyclooxygenase-2 (COX-2), consequent p53 phosphorylation and apoptosis. RV-induced ERK1/2 activation is involved in not only COX-2 expression but also nuclear COX-2 accumulation. NS-398, a COX-2 inhibitor, did not affect ERK1/2 activation, but reduced the nuclear abundance of COX-2 protein and the formation of complexes of nuclear COX-2 and activated ERK1/2 that are required for p53-dependent apoptosis in RVtreated cells. T 4 inhibited RV-induced nuclear COX-2 and cytosolic pro-apoptotic protein, BcLx-s, accumulation. Furthermore, T 4 inhibited RV-induced apoptosis by interfering with the interaction of nuclear COX-2 and ERK1/2. This effect of T 4 was prevented by tetraiodothyroacetic acid (tetrac), an inhibitor of the binding of thyroid hormone to its integrin receptor. Tetrac did not, in the absence of T 4 , affect induction of apoptosis by RV. Thus, the receptor sites on aVb3 for RV and thyroid hormone are discrete and activate ERK1/2-dependent downstream effects on apoptosis that are distinctive.

Recent evidence suggests that the thyroid hormone L-thyroxine (T 4 ) stimulates growth of cancer cells via a plasma membrane receptor on integrin A V B 3 . The contribution of this recently described receptor for thyroid hormone and receptor-based stimulation of cellular mitogen-activated protein kinase [MAPK; extracellular signal-regulated kinase 1/2 (ERK1/2)] activity, to enhancement of cell proliferation by thyroid hormone was quantitated functionally and by immunologic means in three glioma cell lines exposed to T 4 . At concentrations of 1 to 100 nmol/L, T 4 caused proliferation of C6, F98, and GL261 cells, measured by accumulation of proliferating cell nuclear antigen (PCNA) and radiolabeled thymidine incorporation. This effect was inhibited by the T 4 analogue, tetraiodothyroacetic acid, and by an A V B 3 RGD recognition site peptide, both of which block T 4 binding to integrin A V B 3 but are not agonists. Activation of MAPK by T 4 was similarly inhibited by tetraiodothyroacetic acid and the RGD peptide. The thyroid hormone 3,5,3 ¶-triiodo-L-thyronine (T 3 ) and T 4 were equipotent stimulators of PCNA accumulation in C6, F98, and GL261 cells, but physiologic concentrations of T 3 are 50-fold lower than those of T 4 . In conclusion, our studies suggest that glioblastoma cells are thyroid hormone dependent and provide a molecular basis for recent clinical observations that induction of mild hypothyroidism may improve duration of survival in glioblastoma patients. The present experiments infer a novel cell membrane receptor-mediated basis for the growth-promoting activity of thyroid hormone in such tumors and suggest new therapeutic approaches to the treatment of patients with glioblastoma. (Cancer Res 2006; 66(14): 7270-5)

We have demonstrated in cultured cells that L-thyroxine (T 4 ) 1 can nongenomically activate signal transduction proteins such as mitogen-activated protein kinase (MAPK) (1) and, through serine phosphorylation by MAPK, can enhance the activity of several nuclear transactivator proteins. Among the latter are the signal transducer and activator of transcription (STAT) proteins that mediate growth factor (2) and cytokine (1, 3) signals. This T 4 effect is initiated by a G protein-coupled receptor in the plasma membrane (1) and has been observed in cells that contain endogenous nuclear thyroid hormone receptor (TR), such as 293T cells and human skin fibroblasts (BG-9), and in cells which are devoid of functional TR (CV-1 and HeLa (4)).In contrast, genomic actions of thyroid hormone require binding of the hormone, predominantly 3,5,3Ј-triiodo-L-thyronine (T 3 ), by specific receptors in the cell nucleus. T 3 -liganded TR may bind as a monomer or a homo-or heterodimer with retinoid X receptor to thyroid hormone response elements in the regulatory upstream regions of specific hormone-responsive genes (5-7). In the absence of T 3 , TR exists in the transcriptionally inactive (repressed) state. This state is imposed by the binding to unliganded TR of the co-repressor proteins, SMRT (silencing mediator of retinoid and thyroid hormone receptors) and NCoR (nuclear co-repressor) (8). SMRT binding to TR has been localized to the hinge region of the receptor (amino acids 211-240) (9). Binding of T 3 to TR results in dissociation of co-repressor proteins from TR and the recruitment of activator proteins that facilitate enhanced transcriptional activity of the receptor (10).Serine phosphorylation of TR isoforms has been described by several laboratories (11)(12)(13)(14)(15). In such studies phosphorylation has been inferred from stimulation of the activity of cellular cAMP-dependent protein kinase (PKA) (11, 12), a serine/threonine kinase, or from serine kinase inhibition or phosphatase inhibition in treated cells (14, 16). There have been several results of such phosphorylation in these model systems. For example, serine phosphorylation of TR␣1 has been shown to decrease TR monomer binding to DNA (11). TR␤1 is selectively stabilized against protease degradation by serine phosphorylation and transcriptional activity of the receptor is significantly increased (14,15). Leitman et al. (12) have also shown increased transcriptional activity of TR␤1 in response to phosphorylation. Comparing phosphorylatable and nonphosphorylatable forms of TR␣2, Katz et al. (17) concluded that serine phosphorylation of the receptor isoform decreased its ability to heterodimerize with retinoid X receptor at a thyroid hormone response element site. Using a serine/threonine kinase inhibitor, H7, Jones et al. (16) showed a reduction in T 3 -induced transcriptional activity of both TR␣1 and TR␤1. In some of these studies (12), the results were highly cell line-specific.The mechanism by which serine phosphorylation of TR␤1 is achieved is unclear, aside ...

Resveratrol is a naturally occurring polyphenol, which causes apoptosis in cultured cancer cells. We describe a cell surface resveratrol receptor on the extracellular domain of hetero-dimeric alphaVbeta3 integrin in MCF-7 human breast cancer cells. This receptor is linked to induction by resveratrol of extracellular-regulated kinases 1 and 2 (ERK1/2)- and serine-15-p53-dependent phosphorylation leading to apoptosis. The integrin receptor is near the Arg-Gly-Asp (RGD) recognition site on the integrin; an integrin-binding RGD peptide inhibits induction by resveratrol of ERK1/2- and p53-dependent apoptosis. Antibody (Ab) to integrin alphaVbeta3, but not to alphaVbeta5, inhibits activation by resveratrol of ERK1/2 and p53 and consequent apoptosis in estrogen receptor-alpha (ERalpha) positive MCF-7, and ERalpha-negative MDA-MB231 cells. Resveratrol is displaced from the purified integrin by an RGD, but not RGE, peptide, and by alphaVbeta3 integrin-specific Ab. Resveratrol action is blocked by siRNAbeta3, but not by siRNAalphaV. [14C]-Resveratrol binds to commercially purified integrin alphaVbeta3 and to alphaVbeta3 prepared from MCF-7 cells; binding of [14C]-resveratrol to the beta3, but not to the alphaV monomer, is displaced by unlabeled resveratrol. In conclusion, binding of resveratrol to integrin alphaVbeta3, principally to the beta3 monomer, is essential for transduction of the stilbene signal into p53-dependent apoptosis of breast cancer cells.

No abstract

Cyclooxygenase-2 (COX-2) is antiapoptotic and is implicated in tumorigenesis. Recent reports, however, have also ascribed a proapoptotic action to inducible COX-2. We show here for the first time that a stilbene, resveratrol, induces nuclear accumulation of COX-2 protein in human breast cancer MCF-7 and MDA-MB-231 cell cultures. The induction of COX-2 accumulation by resveratrol is mitogen-activated protein kinase (MAPK; extracellular signal -regulated kinase 1/2)-and activator protein 1-dependent. Nuclear COX-2 in resveratrol-treated cells colocalizes with Ser 15 -phosphorylated p53 and with p300, a coactivator for p53-dependent gene expression. The interaction of COX-2, p53, and p300, as well as resveratrol-induced apoptosis, was inhibited by a MAPK activation inhibitor, PD98059. A specific inhibitor of COX-2, NS398, and small interfering RNA knockdown of COX-2 were associated with reduced p53 phosphorylation and consequent decrease in p53-dependent apoptosis in resveratrol-treated cells. We conclude that nuclear accumulation of COX-2 can be induced by resveratrol and that the COX has a novel intranuclear colocalization with Ser 15