The SREBP-dependent regulation of cyclin D1 coordinates cell proliferation and lipid synthesis.

The sterol regulatory-element binding protein (SREBP) family of transcription factors regulates cholesterol, fatty acid, and triglyceride synthesis and metabolism. However, they are also targeted by the ubiquitin ligase Fbw7, a major tumor suppressor, suggesting that they could regulate cell growth. Indeed, enhanced lipid synthesis is a hallmark of many human tumors. Thus, the SREBP pathway has recently emerged as a potential target for cancer therapy. We have previously demonstrated that one of these transcription factors, SREBP1, is stabilized and remains associated with target promoters during mitosis, suggesting that the expression of these target genes could be important as cells enter G1 and transcription is restored. Activation of cyclin D-cdk4/6 complexes is critical for the phosphorylation and inactivation of the retinoblastoma protein (Rb) family of transcriptional repressors and progression through the G1 phase of the cell cycle. Importantly, the cyclin D-cdk4/6-Rb regulatory axis is frequently dysregulated in human cancer. In the current manuscript, we demonstrate that SREBP1 activates the expression of cyclin D1, a coactivator of cdk4 and cdk6, by binding to an E-box in the cyclin D1 promoter. Consequently, inactivation of SREBP1 in human liver and breast cancer cell lines reduces the expression of cyclin D1 and attenuates Rb phosphorylation. Rb phosphorylation in these cells can be rescued by restoring cyclin D1 expression. On the other hand, expression of active SREBP1 induced the expression of cyclin D1 and increased the phosphorylation of Rb in a manner dependent on cyclin D1 and cdk4/6 activity. Inactivation of SREBP1 resulted in reduced expression of cyclin D1, attenuated phosphorylation of Rb, and reduced proliferation. Inactivation of SREBP1 also reduced the insulin-dependent regulation of the cyclin D1 gene. At the same time, SREBP1 is known to play an important role in supporting lipid synthesis in cancer cells. Thus, we propose that the SREBP1-dependent regulation of cyclin D1 coordinates cell proliferation with the enhanced lipid synthesis required to support cell growth.

Loss of the Fbw7 tumor suppressor rewires cholesterol metabolism in cancer cells leading to activation of the PI3K-AKT signalling axis.

The sterol regulatory-element binding proteins (SREBPs) are transcription factors controlling cholesterol and fatty acid synthesis and metabolism. There are three SREBP proteins, SREBP1a, SREBP1c and SREBP2, with SREBP1a being the strongest transcription factor. The expression of SREBP1a is restricted to rapidly proliferating cells, including cancer cells. The SREBP proteins are translated as large, inactive precursors bound to the endoplasmic reticulum (ER) membranes. These precursors undergo a two-step cleavage process that releases the amino terminal domains of the proteins, which translocate to the nucleus and function as transcription factors. The nuclear forms of the SREBPs are rapidly degraded by the ubiquitin-proteasome system in a manner dependent on the Fbw7 ubiquitin ligase. Consequently, inactivation of Fbw7 results in the stabilization of active SREBP1 and SREBP2 and enhanced expression of target genes. We report that the inactivation of Fbw7 in cancer cells blocks the proteolytic maturation of SREBP2. The same is true in cells expressing a cancer-specific loss-of-function Fbw7 protein. Interestingly, the activation of SREBP2 is restored in response to cholesterol depletion, suggesting that Fbw7-deficient cells accumulate cholesterol. Importantly, inactivation of SREBP1 in Fbw7-deficient cells also restores the cholesterol-dependent regulation of SREBP2, suggesting that the stabilization of active SREBP1 molecules could be responsible for the blunted activation of SREBP2 in Fbw7-deficient cancer cells. We suggest that this could be an important negative feedback loop in cancer cells with Fbw7 loss-of-function mutations to protect these cells from the accumulation of toxic levels of cholesterol and/or cholesterol metabolites. Surprisingly, we also found that the inactivation of Fbw7 resulted in the activation of AKT. Importantly, the activation of AKT was dependent on SREBP1 and on the accumulation of cholesterol. Thus, we suggest that the loss of Fbw7 rewires lipid metabolism in cancer cells to support cell proliferation and survival.

Importance of mesh overlap on hernia recurrence after open umbilical hernia repair with bilayer prosthesis.

BACKGROUND: importance of mesh overlap on recurrence after open umbilical hernia repair has been poorly studied. METHODS: a retrospective cohort study was performed with patients who underwent open umbilical hernia repair with bilayer prosthesis between 2004 and 2015. RESULTS: 1538 patients were included. Fifty patients (3.3%) had a mesh overlap lower than 1 cm. After a mean follow-up of 4.1 years 53 patients (3.5%) developed a recurrence. Recurrence was associated with a mesh overlap smaller than 1 cm (10.2% vs. 3.3%, p = 0.010, OR = 3.3). In the logistic regression model an overlap smaller than 1 cm was not statistically associated with recurrence (OR = 2.5, p = 0.123). Female gender, postoperative complications and prosthesis size were associated with hernia recurrence. CONCLUSIONS: mesh overlap seems to be an important factor for hernia recurrence. A mesh overlap of at least 1 cm should be used until more studies are performed about this issue.

Umbilical Hernia Repair: Analysis After 934 Procedures.

There is a lack of consensus about the surgical management of umbilical hernias. The aim of this study is to analyze the medium-term results of 934 umbilical hernia repairs. In this study, 934 patients with an umbilical hernia underwent surgery between 2004 and 2010, 599 (64.1%) of which were evaluated at least one year after the surgery. Complications, recurrence, and the reoperation rate were analyzed. Complications were observed in 5.7 per cent of the patients. With a mean follow-up time of 35.5 months, recurrence and reoperation rates were 3.8 per cent and 4.7 per cent, respectively. A higher percentage of female patients (60.9 % vs 29 %, P = 0.001) and a longer follow-up time (47.4 vs 35 months, P = 0.037) were observed in patients who developed a recurrence. No significant differences were observed between complications and the reoperation rate in patients who underwent Ventralex(®) preperitoneal mesh reinforcement and suture repair; however, a trend toward a higher recurrence rate was observed in patients with suture repair (6.5 % vs 3.2 %, P = 0.082). Suture repair had lower recurrence and reoperation rates in patients with umbilical hernias less than 1 cm. Suture repair is an appropriate procedure for small umbilical hernias; however, for larger umbilical hernias, mesh reinforcement should be considered.

Randomised clinical trial: conventional Lichtenstein vs. hernioplasty with self-adhesive mesh in bilateral inguinal hernia surgery.

PURPOSE: To compare the results of conventional Lichtenstein hernioplasty with polypropylene mesh (PLP) with a lightweight self-adhesive mesh (Parietene Progrip®; Covidien, Dublin, Ireland) (PPG) used in patients with bilateral inguinal hernia. METHODS: Randomised clinical trial with 89 patients with a minimum follow-up of 1 year. Every patient had bilateral inguinal hernia and had both prostheses implanted randomly, one on each side. Early postoperative and chronic pain was evaluated using the visual analogue scale. Also recurrence rate and subjective evaluation of patients were analysed. RESULTS: Pain in the early postoperative period was inferior on the side where the self-adhesive mesh had been implanted (6.12 vs. 6.62, p=0.005 during the 1st postoperative day; 2.12 vs. 2.62, p=0.001 during the 7th postoperative day). Differences disappeared with the long-term evaluation (0.71 vs. 0.98, p=0.148 1 year after the surgery). The operative time was significantly shorter on the PPG mesh side (24.37 ± 5.1 in case of the PPG mesh and 29.66 ± 5.6 in case of the PLP mesh, p<0,001). Recurrence occurred in seven patients (7.8%), six of them (6.7%, CI 3.0-14.4) on the PPG mesh side and one (1.1%, CI 0.2-7.8) on the PLP side. These differences were not statistically significant (p=0.125) CONCLUSIONS: Although hernioplasty with self-adhesive mesh reduced early postoperative pain, this reduction was clinically irrelevant and it had no influence on chronic pain. There was a trend towards a higher recurrence rate when self-adhesive meshes were used, and although in this study differences were not statistically significant they should be confirmed in later studies using larger samples. Surgical procedures that do not need fixing sutures are promising, but further studies are needed before they become the gold standard of inguinal hernia repair.

Fbw7 dimerization determines the specificity and robustness of substrate degradation.

The Fbw7 tumor suppressor targets a broad network of proteins for ubiquitylation. Here we show critical functions for Fbw7 dimerization in regulating the specificity and robustness of degradation. Dimerization enables Fbw7 to target substrates through concerted binding to two suboptimal and independent recognition sites. Accordingly, an endogenous dimerization-deficient Fbw7 mutation stabilizes suboptimal substrates. Dimerization increases Fbw7's robustness by preserving its function in the setting of mutations that disable Fbw7 monomers, thereby buffering against pathogenic mutations. Finally, dimerization regulates Fbw7 stability, and this likely involves Fbw7 trans-autoubiquitylation. Our study reveals novel functions of Fbw7 dimerization and an unanticipated complexity in substrate degradation.

The ubiquitin ligase Fbxw7 controls adipocyte differentiation by targeting C/EBPalpha for degradation.

Adipose tissue controls body lipid and energy metabolism, as well as food intake, and abnormalities in adipose function play a central role in diseases such as obesity and type-2 diabetes. Adipocyte differentiation is controlled by a transcriptional cascade involving PPARgamma and members of the C/EBP family of transcription factors. Here, we demonstrate that C/EBPalpha is targeted for degradation by the ubiquitin ligase Fbxw7 in a phosphorylation-dependent manner. Importantly, inactivation of Fbxw7 is sufficient to convert mouse preadipocytes into mature adipocytes in a manner dependent on C/EBPalpha. In addition, inactivation of Fbxw7 promotes adipocyte differentiation of human adult stem cells. Taken together, our results suggest that Fbxw7 is a negative regulator of adipogenesis by targeting C/EBPalpha for degradation. This notion is supported by the observation that the expression of Fbxw7 is down-regulated during adipocyte differentiation, resulting in the accumulation of proadipogenic proteins such as C/EBPalpha. Thus, Fbxw7 could be an important regulator of energy and lipid metabolism.

A phosphorylation cascade controls the degradation of active SREBP1.

Sterol regulatory element-binding proteins (SREBPs) are a family of transcription factors that regulates cholesterol and lipid metabolism. The active forms of these transcription factors are targeted by a number of post-translational modifications, including phosphorylation. Phosphorylation of Thr-426 and Ser-430 in SREBP1a creates a docking site for the ubiquitin ligase Fbw7, resulting in the degradation of the transcription factor. Here, we identify a novel phosphorylation site in SREBP1a, Ser-434, which regulates the Fbw7-dependent degradation of SREBP1. We demonstrate that both SREBP1a and SREBP1c are phosphorylated on this residue (Ser-410 in SREBP1c). Importantly, we demonstrate that the mature form of endogenous SREBP1 is phosphorylated on Ser-434. Glycogen synthase kinase-3 phosphorylates Ser-434, and the phosphorylation of this residue is attenuated in response to insulin signaling. Interestingly, phosphorylation of Ser-434 promotes the glycogen synthase kinase-3-dependent phosphorylation of Thr-426 and Ser-430 and destabilizes SREBP1. Consequently, mutation of Ser-434 blocks the interaction between SREBP1 and Fbw7 and attenuates Fbw7-dependent degradation of SREBP1. Importantly, insulin fails to enhance the levels of mature SREBP1 in cells lacking Fbw7. Thus, the degradation of mature SREBP1 is controlled by cross-talk between multiple phosphorylated residues in its C-terminal domain and the phosphorylation of Ser-434 could function as a molecular switch to control these processes.

An integrated gene regulatory network controls stem cell proliferation in teeth.

Epithelial stem cells reside in specific niches that regulate their self-renewal and differentiation, and are responsible for the continuous regeneration of tissues such as hair, skin, and gut. Although the regenerative potential of mammalian teeth is limited, mouse incisors grow continuously throughout life and contain stem cells at their proximal ends in the cervical loops. In the labial cervical loop, the epithelial stem cells proliferate and migrate along the labial surface, differentiating into enamel-forming ameloblasts. In contrast, the lingual cervical loop contains fewer proliferating stem cells, and the lingual incisor surface lacks ameloblasts and enamel. Here we have used a combination of mouse mutant analyses, organ culture experiments, and expression studies to identify the key signaling molecules that regulate stem cell proliferation in the rodent incisor stem cell niche, and to elucidate their role in the generation of the intrinsic asymmetry of the incisors. We show that epithelial stem cell proliferation in the cervical loops is controlled by an integrated gene regulatory network consisting of Activin, bone morphogenetic protein (BMP), fibroblast growth factor (FGF), and Follistatin within the incisor stem cell niche. Mesenchymal FGF3 stimulates epithelial stem cell proliferation, and BMP4 represses Fgf3 expression. In turn, Activin, which is strongly expressed in labial mesenchyme, inhibits the repressive effect of BMP4 and restricts Fgf3 expression to labial dental mesenchyme, resulting in increased stem cell proliferation and a large, labial stem cell niche. Follistatin limits the number of lingual stem cells, further contributing to the characteristic asymmetry of mouse incisors, and on the basis of our findings, we suggest a model in which Follistatin antagonizes the activity of Activin. These results show how the spatially restricted and balanced effects of specific components of a signaling network can regulate stem cell proliferation in the niche and account for asymmetric organogenesis. Subtle variations in this or related regulatory networks may explain the different regenerative capacities of various organs and animal species.

SREBP in signal transduction: cholesterol metabolism and beyond.

The last few years have seen important advances in defining the mechanisms that cells use to monitor changes in cholesterol levels and regulate lipid metabolism. This work has unraveled a feedback system that enables cholesterol and certain sterol intermediates to regulate the proteolysis and transport of specific membrane proteins. The sterol regulatory element-binding protein (SREBP) family of transcription factors is at the center of this feedback system. These membrane-embedded proteins are activated by ER-to-Golgi transport followed by limited proteolysis. In addition, both the activation of the SREBPs and the stability of the rate-limiting enzyme in cholesterol synthesis are regulated by the ubiquitin-proteasome system in a sterol-dependent manner. The ubiquitin-proteasome system also regulates the degradation of active SREBPs. Recent work also highlights the important role of this regulatory system in several organisms, ranging from yeast to humans. In addition, the SREBP pathway has been found to regulate a diverse set of cellular processes, including phagocytosis, cell cycle progression, oxygen sensing and survival in response to bacterial infection. These advances illustrate the wide-ranging roles that SREBPs and membrane biogenesis have in cell biology.

Bioluminescence imaging of mitochondrial Ca2+ dynamics in soma and neurites of individual adult mouse sympathetic neurons.

Changes in the cytosolic Ca(2+) concentration ([Ca(2+)](c)) are essential for triggering neurotransmitter release from presynaptic nerve terminals. Calcium-induced Ca(2+) release (CICR) from the endoplasmic reticulum (ER) may amplify the [Ca(2+)](c) signals and facilitate neurotransmitter release in sympathetic neurons. In adrenal chromaffin cells, functional triads are formed by voltage-operated Ca(2+) channels (VOCCs), CICR sites and mitochondria. In fact, mitochondria take up most of the Ca(2+) load entering the cells and are essential for shaping [Ca(2+)](c) signals and exocytosis. Here we have investigated the existence of such functional triads in sympathetic neurons. The mitochondrial Ca(2+) concentration ([Ca(2+)](m)) in soma and neurites of individual mouse superior cervical ganglion (SCG) neurons was monitored by bioluminescence imaging of targeted aequorins. In soma, Ca(2+) entry through VOCCs evoked rapid, near millimolar [Ca(2+)](m) increases in a subpopulation of mitochondria containing about 40% of the aequorin. Caffeine evoked a similar [Ca(2+)](m) increase in a mitochondrial pool containing about 30% of the aequorin and overlapping with the VOCC-sensitive pool. These observations suggest the existence of functional triads similar to the ones described in chromaffin cells. In neurites, mitochondria were able to buffer [Ca(2+)](c) increases resulting from activation of VOCCs but not those mediated by caffeine-induced Ca(2+) release from the ER. The weaker Ca(2+) buffering by mitochondria in neurites could contribute to facilitate Ca(2+)-induced exocytosis at the presynaptic sites.

Cdk1/cyclin B-mediated phosphorylation stabilizes SREBP1 during mitosis.

Members of the sterol regulatory element-binding protein (SREBP) family of transcription factors control the biosynthesis of cholesterol and other lipids, and lipid synthesis is critical for cell growth and proliferation. We recently found that the mature forms of SREBP1a and SREBP1c are hyperphosphorylated in mitotic cells, giving rise to a phosphoepitope recognized by the mitotic protein monoclonal-2 (MPM-2) antibody. In addition, we found that mature SREBP1 was stabilized in a phosphorylation-dependent manner during mitosis. We have now mapped the major MPM-2 epitope to a serine residue, S439, in the C terminus of mature SREBP1. Using phosphorylation-specific antibodies, we demonstrate that endogenous SREBP1 is phosphorylated on S439 specifically during mitosis. Mature SREBP1 interacts with the Cdk1/cyclin B complex in mitotic cells and we demonstrate that Cdk1 phosphorylates S439, both in vitro and in vivo. Our results suggest that Cdk1-mediated phosphorylation of S439 stabilizes mature SREBP1 during mitosis, thereby preserving a critical pool of active transcription factors to support lipid synthesis. Taken together with our previous work, the current study suggests that SREBP1 may provide a link between lipid synthesis, proliferation and cell growth. This hypothesis was supported by our observation that siRNA-mediated inactivation of SREBP1 arrested cells in the G(1) phase of the cell cycle, thereby attenuating cell growth.

Phosphorylation and ubiquitination of the transcription factor sterol regulatory element-binding protein-1 in response to DNA binding.

Members of the sterol regulatory element-binding protein (SREBP) family of transcription factors control cholesterol and lipid metabolism and play critical roles during adipocyte differentiation. The transcription factor SREBP1 is degraded by the ubiquitin-proteasome system following phosphorylation of Thr426 and Ser430 in its phosphodegron. We now demonstrate that the glycogen synthase kinase (GSK)-3beta-dependent phosphorylation of these residues in SREBP1 is enhanced in response to specific DNA binding. DNA binding enhances the direct interaction between the C-terminal domain of SREBP1 and GSK-3beta. Accordingly, we demonstrate that GSK-3beta is recruited to the promoters of SREBP target genes in vivo. As a result of the phosphorylation of Thr426 and Ser430, the ubiquitin ligase Fbw7 is recruited to SREBP molecules associated with target promoters. Using a reconstituted ubiquitination system, we demonstrate that Fbw7-mediated ubiquitination of SREBP1 is dependent on its DNA binding activity. Thus, DNA binding could provide a mechanistic link between the phosphorylation, ubiquitination, and degradation of active transcription factors.

Control of lipid metabolism by phosphorylation-dependent degradation of the SREBP family of transcription factors by SCF(Fbw7).

The sterol regulatory element binding protein (SREBP) family of transcription factors controls cholesterol and lipid metabolism. The nuclear forms of these proteins are rapidly degraded by the ubiquitin-proteasome pathway, but the signals and factors required for this are unknown. Here, we identify a phosphodegron in SREBP1a that serves as a recognition motif for the SCF(Fbw7) ubiquitin ligase. Fbw7 interacts with nuclear SREBP1a and enhances its ubiquitination and degradation in a manner dependent on the phosphorylation of T426 and S430 by GSK-3. Fbw7 also degrades nuclear SREBP1c and SREBP2, and inactivation of endogenous Fbw7 results in stabilization of nuclear SREBP1 and -2, enhanced expression of SREBP target genes, enhanced synthesis of cholesterol and fatty acids, and enhanced receptor-mediated uptake of LDL. Thus, our results suggest that Fbw7 may be a major regulator of lipid metabolism through control of the phosphorylation-dependent degradation of the SREBP family of transcription factors.

Hyperphosphorylation regulates the activity of SREBP1 during mitosis.

The sterol regulatory element-binding protein (SREBP) family of transcription factors controls the biosynthesis of cholesterol and other lipids, and lipid synthesis is critical for cell growth and proliferation. We were, therefore, interested in the expression and activity of SREBPs during the cell cycle. We found that the expression of a number of SREBP-responsive promoter-reporter genes were induced in a SREBP-dependent manner in cells arrested in G2/M. In addition, the mature forms of SREBP1a and SREBP1c were hyperphosphorylated in mitotic cells, giving rise to a phosphoepitope recognized by the mitotic protein monoclonal-2 (MPM-2) antibody. In contrast, SREBP2 was not hyperphosphorylated in mitotic cells and was not recognized by the MPM-2 antibody. The MPM-2 epitope was mapped to the C terminus of mature SREBP1, and the mitosis-specific hyperphosphorylation of SREBP1 depended on this domain of the protein. The transcriptional and DNA-binding activity of SREBP1 was enhanced in cells arrested in G2/M, and these effects depended on the C-terminal domain of the protein. In part, these effects could be explained by our observation that mature SREBP1 was stabilized in G2/M. In agreement with these observations, we found that the synthesis of cholesterol was increased in G2/M-arrested cells. Thus, our results demonstrate that the activity of mature SREBP1 is regulated by phosphorylation during the cell cycle, suggesting that SREBP1 may provide a link between lipid synthesis, proliferation, and cell growth.

Bioluminescence imaging of nuclear calcium oscillations in intact pancreatic islets of Langerhans from the mouse.

The stimulus-secretion coupling for insulin secretion by pancreatic beta cells in response to high glucose involves synchronic cytosolic calcium oscillations driven by bursting electrical activity. Calcium inside organelles can regulate additional functions, but analysis of subcellular calcium signals, specially at the single cell level, has been hampered for technical constrains. Here we have monitored nuclear calcium oscillations by bioluminescence imaging of targeted aequorin in individual cells within intact islets of Langerhans as well as in the whole islet. We find that glucose generates a pattern of nuclear calcium oscillations resembling those found in the cytosol. Some cells showed synchronous nuclear calcium oscillations suggesting that the islet of Langerhans may also regulate the activation of Ca(2+)-responsive nuclear processes, such as gene transcription, in a coordinated, synchronic manner. The nuclear Ca(2+) oscillations are due to bursting electrical activity and activation of plasma membrane voltage-gated Ca(2+) channels with little or no contribution of calcium release from the intracellular Ca(2+) stores. Irregularities in consumption of aequorins suggests that depolarization may generate formation of steep Ca(2+) gradients in both the cytosol and the nucleus, but further research is required to investigate the role of such high [Ca(2+)] microdomains.

The nature of the rate-limiting steps in the refolding of the cofactor-dependent protein aspartate aminotransferase.

The refolding of mitochondrial aspartate aminotransferase (mAAT; EC 2.6.1.1) has been studied following unfolding in 6 m guanidine hydrochloride for different periods of time. Whereas reactivation of equilibrium-unfolded mAAT is sigmoidal, reactivation of the short term unfolded protein displays a double exponential behavior consistent with the presence of fast and slow refolding species. The amplitude of the fast phase decreases with increasing unfolding times (k approximately 0.75 min(-1) at 20 degrees C) and becomes undetectable at equilibrium unfolding. According to hydrogen exchange and stopped-flow intrinsic fluorescence data, unfolding of mAAT appears to be complete in less than 10 s, but hydrolysis of the Schiff base linking the coenzyme pyridoxal 5'-phosphate (PLP) to the polypeptide is much slower (k approximately 0.08 min(-1)). This implies the existence in short term unfolded samples of unfolded species with PLP still attached. However, since the disappearance of the fast refolding phase is about 10-fold faster than the release of PLP, the fast refolding phase does not correspond to folding of the coenzyme-containing molecules. The fast refolding phase disappears more rapidly in the pyridoxamine and apoenzyme forms of mAAT, both of which lack covalently attached cofactor. Thus, bound PLP increases the kinetic stability of the fast refolding unfolding intermediates. Conversion between fast and slow folding forms also takes place in an early folding intermediate. The presence of cyclophilin has no effect on the reactivation of either equilibrium or short term unfolded mAAT. These results suggest that proline isomerization may not be the only factor determining the slow refolding of this cofactor-dependent protein.