b; Université catholique de Louvain, Brussels, Belgium c Eukaryotic elongation factor 2 kinase (eEF2K), an atypical calmodulin-dependent protein kinase, phosphorylates and inhibits eEF2, slowing down translation elongation. eEF2K contains an N-terminal catalytic domain, a C-terminal ␣-helical region and a linker containing several regulatory phosphorylation sites. eEF2K is expressed at high levels in certain cancers, where it may act to help cell survival, e.g., during nutrient starvation. However, it is a negative regulator of protein synthesis and thus cell growth, suggesting that cancer cells may possess mechanisms to inhibit eEF2K under good growth conditions, to allow protein synthesis to proceed. We show here that the mTORC1 pathway and the oncogenic Ras/Raf/MEK/extracellular signal-regulated kinase (ERK) pathway cooperate to restrict eEF2K activity. We identify multiple sites in eEF2K whose phosphorylation is regulated by mTORC1 and/or ERK, including new ones in the linker region. We demonstrate that certain sites are phosphorylated directly by mTOR or ERK. Our data reveal that glycogen synthase kinase 3 signaling also regulates eEF2 phosphorylation. In addition, we show that phosphorylation sites remote from the N-terminal calmodulin-binding motif regulate the phosphorylation of N-terminal sites that control CaM binding. Mutations in the former sites, which occur in cancer cells, cause the activation of eEF2K. eEF2K is thus regulated by a network of oncogenic signaling pathways.

Hypoxia-inducible factor-1 (HIF-1) is a heterodimeric transcription factor that plays a major role in cellular adaptation to hypoxia. The mechanisms regulating HIF-1 activity occurs at multiple levels in vivo. The HIF-1a subunit is highly sensible to oxygen and is rapidly degraded by the proteasome 26S in normoxia. Activation in hypoxia occurs through a multistep process including inhibition of HIF-1a degradation, but also increase in the transactivation activity of HIF-1. Several data indicate that phosphorylation could play a role in this regulation. In this report, we investigated the role of casein kinase 2 (CK2), an ubiquitous serine/ threonine kinase, in the regulation of HIF-1 activity. Hypoxia was capable of increasing the expression of the b subunit of CK2, of inducing a relocalization of this subunit at the plasma membrane, of inducing nuclear translocation of the a subunit and of increasing CK2 activity. Three inhibitors of this kinase, DRB (5,6-dichloro-1-b-D-ribofuranosyl-benzimidazole), TBB (4,5,6,7-tetrabromotriazole) and apigenin, as well as overexpression of a partial dominant negative mutant of CK2a, were shown to inhibit HIF-1 activity as measured by a reporter assay and through hypoxiainduced VEGF and aldolase expression. This does not occur at the stabilization process since they did not affect HIF-1a protein level. DNA-binding activity was also not inhibited. We conclude that CK2 is an important regulator of HIF-1 transcriptional activity but the mechanism of this regulation remains to be determined. Since HIF-1 plays a major role in tumor angiogenesis and since CK2 has been described to be overexpressed in tumor cells, this new pathway of regulation can be one more way for tumor cells to survive. ' 2005 Wiley-Liss, Inc.Key words: casein kinase 2; HIF-1; neoangiogenesis HIF-1 (hypoxia-inducible factor-1) is a key regulator of cell response to reduced oxygen level. In response to hypoxic conditions, HIF-1 increases the expression of downstream target genes such as erythropoietin (EPO), vascular endothelial growth factor (VEGF) and glycolytic enzymes (aldolase A, enolase-a) and mediates adaptation of cells to decreased oxygen level. 1 The role of HIF-1 in the regulation of tumor growth by hypoxia via the initiation of angiogenesis is certainly the best example of such an adaptive response. 2 Oncogene activation and tumor-suppressor inactivation result in deregulated cellular proliferation. However, most tumors grown larger than 1 mm 3 contain regions of low oxygen tension (hypoxia) due to an imbalance between oxygen supply and consumption. Formation of new blood vessels or ''neoangiogenesis'' is thus essential for further tumor growth. It is also important to allow tumor cell dissemination at distant sites, i.e., metastasis.Several studies using HIF-1 mutant cells have shown that HIF-1 has a profound effect on tumor biology. For example, tumors grown from HIF-1a-defective embryonic stem cells display abnormal vascularity and reduced growth rate. 3 Moreover, HIF-1 is upregulated in a broad r...

Abstract STUDY QUESTION How do elastic matrisome components change during the lifetime of the human ovary? SUMMARY ANSWER The deposition and remodeling of mechanical matrisome components (collagen, elastin, elastin microfibril interface-located protein 1 (EMILIN-1), fibrillin-1 and glycosaminoglycans (GAGs)) that play key roles in signaling pathways related to follicle activation and development evolve in an age- and follicle stage-related manner. WHAT IS KNOWN ALREADY The mechanobiology of the human ovary and dynamic reciprocity that exists between ovarian cells and their microenvironment is of high importance. Indeed, while the localization of primordial follicles in the collagen-rich ovarian cortex offers a rigid physical environment that supports follicle architecture and probably plays a role in their survival, ovarian extracellular matrix (ECM) stiffness limits follicle expansion and hence oocyte maturation, maintaining follicles in their quiescent state. As growing follicles migrate to the medulla of the ovary, they encounter a softer, more pliant ECM, allowing expansion and development. Thus, changes in the rigidity of the ovarian ECM have a direct effect on follicle behavior. Evidence supporting a role for the physical environment in follicle activation was provided in clinical practice by ovarian tissue fragmentation, which promoted actin polymerization and disrupted ovarian Hippo signaling, leading to increased expression of downstream growth factors, promotion of follicle growth and generation of mature oocytes. STUDY DESIGN, SIZE, DURATION We investigated quantitative spatiotemporal changes in collagen, elastin, EMILIN-1, fibrillin-1 and GAGs from prepuberty to menopause, before conducting a closer analysis of the ECM surrounding follicles, from primordial to secondary stages, in both prepubertal and tissue from women of reproductive age. The study included ovarian tissue (cortex) from 68 patients of different ages: prepubertal (n = 16; mean age [±SD]=8 ± 2 years); reproductive (n = 21; mean age [±SD]=27 ± 4 years); menopausal with estrogen-based HRT (n = 7; mean age [±SD]=58 ± 4 years); and menopausal without HRT (n = 24; mean age [±SD]=61 ± 5 years). PARTICIPANTS/MATERIALS, SETTING, METHODS Quantitative investigations of collagen and GAG deposition in ovarian tissue throughout a woman’s lifetime were conducted by analyzing brightfield images. Characteristic features of collagen fiber content were based on polarized light microscopy, since polarized light changes with fiber thickness. To evaluate the deposition and distribution of elastin, fibrillin-1 and EMILIN-1, multiplex immunofluorescence was used on at least three sections from each patient. Image processing and tailored bioinformatic analysis were applied to enable spatiotemporal quantitative evaluation of elastic system component deposition in the human ovary over its lifetime. MAIN RESULTS AND THE ROLE OF CHANCE While collagen levels increased with age, fibrillin-1 and EMILIN-1 declined. Interestingly, collagen and elastin reached their peak in reproductive-age women compared to prepubertal (P < 0.01; P = 0.262) and menopausal subjects with (P = 0.706; P < 0.01) and without (P = 0.987; P = 0.610) HRT, indicating a positive impact of secreted estrogen and hormone treatment on collagen and elastin preservation. Interestingly, HRT appears to affect elastin presence in ovarian tissue, since a significantly higher (P < 0.05) proportion of elastin was detected in biopsies from menopausal women taking HRT compared to those not. Higher GAG levels were found in adult ovaries compared to prepubertal ovaries (P < 0.05), suggesting changes in tissue ultrastructure and elasticity with age. In this context, elevated GAG values are suspected to participate in hampering formation of the fibrillin-1 network (r = −0.2475; P = 0.04687), which explains its decline over time. This decline partially accounts for the decrease in EMILIN-1 (r = 0.4149; P = 0.00059). Closer examination of the ECM surrounding follicles from the primordial to the secondary stage, both before and after puberty, points to high levels of mechanical stress placed on prepubertal follicles compared to the more compliant ECM around reproductive-age follicles, as suggested by the higher collagen levels and lower elastin content detected mainly around primordial (P < 0.0001; P < 0.0001, respectively) and primary (P < 0.0001; P < 0.001, respectively) follicles. Such a stiff niche is nonpermissive to prepubertal follicle activation and growth, and is more inclined to quiescence. LARGE SCALE DATA Not applicable. LIMITATIONS, REASONS FOR CAUTION The duration and form of administered HRT were not considered when studying the menopausal patient group undergoing treatment. Moreover, we cannot exclude interference from other nongynecological medications taken by the study patients on ovarian ECM properties since there is no information in the literature describing the impact of each medication on the ECM. Finally, since the ECM is by definition a very heterogeneous meshwork of proteins, the use of two-dimensional histology could be a limitation. Single time points on fixed tissues could also present limitations, since following ovary dynamics from prepuberty to menopause in the same patient is not feasible. WIDER IMPLICATIONS OF THE FINDINGS From a biomechanical perspective, our study revealed important changes to ECM properties dictating the mechanical features of ovarian tissue, in line with the existing literature. Our findings pave the way for possible therapeutic targets at the ECM level in the context of female fertility and ovarian rejuvenation, such as mechanical stimulation, antifibrotic treatments, and prevention or reversion of elastic ECM degradation. Our study also sheds light on the follicle-specific ECM composition that is dependent on follicle stage and age. These data will prove very useful in designing biomimetic scaffolds and tissue-engineered models like the artificial ovary. Indeed, they emphasize the importance of encapsulating each type of isolated follicle in an appropriate biomaterial that must replicate the corresponding functional perifollicular ECM and respect ovarian tissue heterogeneity in order to guarantee its biomimicry. STUDY FUNDING/COMPETING INTEREST(S) This study was supported by grants from the Fonds National de la Recherche Scientifique de Belgique (FNRS) (C.A.A. is an FRS-FNRS research associate; grant 5/4/150/5 awarded to M.M.D.) and the Université Catholique de Louvain (PhD grant ‘Coopération au développement’ awarded to E.O.). None of the authors have any competing interests to declare.

Cryopreservation of immature testicular tissue (ITT) prior to chemo/radiotherapy is now ethically accepted and is currently the only way to preserve fertility of prepubertal boys about to undergo cancer therapies. So far, three-dimensional culture of testicular cells isolated from prepubertal human testicular tissue was neither efficient nor reproducible to obtain mature spermatozoa, and ITT transplantation is not a safe option when there is a risk of cancer cell contamination of the testis. Hence, generation of testicular organoids (TOs) after cell selection is a novel strategy aimed at restoring fertility in these patients. Here, we created TOs using hydrogels developed from decellularized porcine ITT and compared cell numbers, organization and function to TOs generated in collagen only hydrogel. Organotypic culture of porcine ITT was used as a control. Rheological and mass spectrometry analyses of both hydrogels highlighted differences in terms of extracellular matrix stiffness and composition, respectively. Sertoli cells (SCs) and germ cells (GCs) assembled into seminiferous tubule-like structures delimited by a basement membrane while Leydig cells (LCs) and peritubular cells localized outside. TOs were maintained for 45 days in culture and secreted stem cell factor and testosterone demonstrating functionality of SCs and LCs, respectively. In both TOs GC numbers decreased and SC numbers increased. However, LC numbers decreased significantly in the collagen hydrogel TOs (p < 0.05) suggesting a better preservation of growth factors within TOs developed from decellularized ITT and thus a better potential to restore the reproductive capacity.