Effect of treadmill exercise and bone marrow stromal cell engraftment on activation of BDNF-ERK-CREB signaling pathway in the crushed sciatic nerve

The effect of combined approach of exercise training and bone marrow stromal cell (BMSC) engraftment on activation of brain-derived neurotrophic factor (BDNF)-extracellular signal-regulated kinase 1 and 2 (ERK1/2)-cyclic adenosine monophosphate response element-binding protein (CREB) signaling pathway after sciatic nerve injury (SNI) was investigated. Sixty male Sprague-Dawley rats divided into the normal control, nonexercise (NEX), exercise training (EX), BMSC transplantation (TP), and exercise training+BMSC transplantation (EX+TP) groups 4 weeks after SCI. Exercise training was carried out on the treadmill device at 5–10 m/min for 20 min for 4 weeks. Single dose of 5× 106 harvested BMSC was injected into the injury area of the injured sciatic nerve. In order to evaluate induction levels of BDNF-ERK1/2-CREB signaling molecules in the whole cell and nuclear cell lysates of the injured sciatic nerve, we applied Western blot analysis. BDNF was significantly increased only in EX+TP compared to NEX, EX, and TP groups. Phosphoinositide-dependent kinase-1 was more increased in EX, TP, and EX+TP groups than NEX group, but EX+TP group showed the most upregulation of phosphorylated protein kinase B compared to other groups. In addition, in the whole cell lysate, phosphorylated ERK1/2, but not activating transcription factor-3 (ATF-3) and phosphorylated CREB, was significantly increased in TP and EX+TP groups. In the nuclear cell lysate, ATF-3 and phosphorylated CREB were strongly activated in EX+TP group compared to EX group. Regular exercise training combined with BMSC engraftment would seem to be more effective in controlling activation of regeneration-related signaling pathway after SNI.

Zinc-mediated activation of CREB pathway in proliferation of pulmonary artery smooth muscle cells in pulmonary hypertension

Background Transcription factor CREB is involved in the development of pulmonary hypertension (PH). However, little is known about the role and regulatory signaling of CREB in PH. Methods A series of techniques, including bioinformatics methods, western blot, cell proliferation and luciferase reporter assay were used to perform a comprehensive analysis of the role and regulation of CREB in proliferation of pulmonary artery smooth muscle cells (PASMCs) in PH. Results Using bioinformatic analysis of the differentially expressed genes (DEGs) identified in the development of monocrotaline (MCT)- and hypoxia-induced PH, we found the overrepresentation of CRE-containing DEGs. Western blot analysis revealed a sustained increase in total- and phosphorylated-CREB in PASMCs isolated from rats treated with MCT. Similarly, an enhanced and prolonged serum-induced CREB phosphorylation was observed in hypoxia-pretreated PASMCs. The sustained CREB phosphorylation in PASMCs may be associated with multiple protein kinases phosphorylated CREB. Additionally, hierarchical clustering analysis showed reduced expression of the majority of CREB phosphatases in PH, including regulatory subunits of PP2A, Ppp2r2c and Ppp2r3a. Cell proliferation analysis showed increased PASMCs proliferation in MCT-induced PH, an effect relied on CREB-mediated transcriptional activity. Further analysis revealed the raised intracellular labile zinc possibly from ZIP12 was associated with reduced phosphatases, increased CREB-mediated transcriptional activity and PASMCs proliferation. Conclusions CREB pathway was overactivated in the development of PH and contributed to PASMCs proliferation, which was associated with multiple protein kinases and/or reduced CREB phosphatases and raised intracellular zinc. Thus, this study may provide a novel insight into the CREB pathway in the pathogenesis of PH.

CREB signaling activity correlates with differentiation and survival in medulloblastoma

While there has been significant progress in the molecular characterization of the childhood brain cancer medulloblastoma, the tumor proteome remains less explored. However, it is important to obtain a complete understanding of medulloblastoma protein biology, since interactions between proteins represent potential new drug targets. Using previously generated phosphoprotein signaling-profiles of a large cohort of primary medulloblastoma, we discovered that phosphorylation of transcription factor CREB strongly correlates with medulloblastoma survival and associates with a differentiation phenotype. We further found that during normal cerebellar development, phosphorylated CREB was selectively expressed in differentiating cerebellar granule neuron progenitor (CGNP) cells. In line, we observed increased differentiation in CGNPs treated with Forskolin, Bmp6 and Bmp12 (Gdf7), which induce CREB phosphorylation. Lastly, we demonstrated that inducing CREB activation via PKA-mediated CREB signaling, but not Bmp/MEK/ERK mediated signalling, enhances medulloblastoma cell sensitivity to chemotherapy.

Detection of Key Proteins in Obstetric Preeclampsia by Nanocomposites Using Gold Nanoparticles/ DNA/Methylene Blue

As a nuclear transcription factor, cAMP response element-binding protein (CREB) can be used in gene transcription regulation, apoptosis regulation, immune response and other life activities. However, the biological characteristics of CREB are controlled by its phosphorylation level. The surface of gold electrode is modified by DNA sequence of capture probe containing specific and high affinity binding sites of CREB. Then, the methylene blue (MB) solution is added into the gold nanoparticle/DNA complex. The composite nanomaterials, namely gold nanoparticles/DNA/MB, are obtained by inserting MB into DNA molecules. The nanomaterials are used as signal markers, and the phosphorylation sites are specifically labeled by Zr4+-mediated nanocomposite beacons. It is used to detect the phosphorylation level of CREB in placenta of preeclampsia pregnant women. In the experiment, the phosphorylated CREB can be fully modified on the surface of gold nanoprobe electrode after incubating for 130 min. The optimal incubation time between nanocomposite beacon and targeted protein electrode is 60 min. Based on this method, the development trend of CREB phosphorylation level is obtained. Comparing the phosphorylation level of CREB in preeclampsia pregnant women and normal pregnant women, it can be seen that the phosphorylation level of CREB in preeclampsia pregnant women is significantly increased. The designed nanocomposites have good sensitivity and specificity for the phosphorylation level of CREB, which can help to analyze and study the protein phosphorylation level of patients under physiological and pathological conditions.

Abstract 15137: β2-adrenergic Signaling Promotes IL-6 Production From Cardiac Fibroblasts Through CREB/Arid5a Pathway and Induces Cardiac Hypertrophy via Paracrine System

Background: β-adrenergic receptor (βAR) is involved in cardiac inflammation and hypertrophy. It has been revealed that βAR stimulation induces cardiac hypertrophy not only directly but also indirectly, for example, via cardiac fibroblasts (CFs). Interestingly, βAR signaling promotes the production of interleukin (IL)-6 from CFs. However, it has not been clarified the relationship between IL-6 released from CFs and cardiac hypertrophy after βAR stimulation. Objective: To elucidate the mechanisms by which βAR signaling promotes the production of IL-6 in CFs and the effects of CFs-secreted IL-6 on cardiac hypertrophy. Methods: CFs were isolated from adult mice. The expression of mRNA was measured by quantitative RT-PCR and the secretion of IL-6 into the medium was quantified by ELISA. Cardiac hypertrophy was assessed by echocardiography, cross-sectional area or cell surface area of cardiomyocytes. Results: RT-PCR revealed that β2AR and β3AR, but not β1AR, were mainly expressed in CFs. Salbutamol (SAL), a selective β2AR agonist, increased IL-6 mRNA by 20-fold, whereas CL-316243, a selective β3AR agonist, did only negligibly. Moreover, SAL, not CL-31243, upregulated the mRNA expression of AT-rich interaction domain 5A (Arid5a), an IL-6 mRNA stabilizing factor, by 4 times. CFs from Arid5a -null mice exhibited reduced expression of IL-6 mRNA and protein, compared with wild-type (WT) CFs, with or without SAL. SAL phosphorylated CREB, suggesting that SAL activated CREB pathway. The compound 666-15, a CREB inhibitor, suppressed SAL-induced IL-6 and Arid5a upregulation. Importantly, WT and IL-6 -null mice were continuously treated with isoproterenol (ISO), a non-selective βAR agonist, for 2 weeks. Cardiac dilatation and cardiomyocyte enlargement were induced by chronic ISO treatment in WT mice, but to lesser extent in IL-6 -null mice. Finally, conditioned media from WT CFs treated with ISO enlarged cultured neonatal rat cardiomyocytes by 10%, while not those from IL-6 -null CFs. Conclusion: β2AR signaling induced IL-6 via CREB/Arid5a axis in CFs. Moreover, CFs-secreted IL-6 contributed to the enlargement of cardiomyocytes through paracrine signaling. β2AR signaling in CFs could be a therapeutic target against cardiac hypertrophy.

CREB is Required in Excitatory Neurons in the Forebrain to Sustain Wakefulness

The molecular and intracellular signaling processes that control sleep and wake states remain largely unknown. A consistent observation is that the cyclic-AMP response element binding protein (CREB), an activity-dependent transcription factor, is differentially activated during sleep and wakefulness. CREB is phosphorylated by the cyclic AMP/protein kinase A (cAMP/PKA) signaling pathway as well as other kinases, and phosphorylated CREB (pCREB) promotes transcription of target genes. Genetic studies in flies and mice suggest that CREB signaling influences sleep/wake states by promoting and stabilizing wakefulness. However, it remains unclear where in the brain CREB is required to drive wakefulness. In rats, CREB phosphorylation increases in the cerebral cortex during wakefulness and decreases during sleep, but it is not known if this change is functionally relevant to the maintenance of wakefulness. Here, we used the cre/lox system to conditionally delete CREB in the forebrain and in the locus coereleus (LC), two regions known to be important for the production of arousal and wakefulness. We used polysomnography to measure sleep/wake levels and sleep architecture in conditional CREB mutant mice and control littermates. We found that forebrain-specific deletion of CREB decreased wakefulness and increased non-rapid eye movement (NREM) sleep. Mice lacking CREB in the forebrain were unable sustain normal periods of wakefulness. On the other hand, deletion of CREB from LC neurons did not change sleep/wake levels or sleep/wake architecture. Taken together, these results suggest that CREB is required in neurons within the forebrain but not in the LC to promote and stabilize wakefulness.

MLL3/MLL4-Associated PAGR1 Regulates Adipogenesis by Controlling Induction of C/EBPβ and C/EBPδ

ABSTRACT Transcription factors C/EBPβ and C/EBPδ are induced within hours after initiation of adipogenesis in culture. They directly promote the expression of master adipogenic transcription factors peroxisome proliferator-activated receptor γ (PPARγ) and C/EBPα and are required for adipogenesis in vivo. However, the mechanism that controls the induction of C/EBPβ and C/EBPδ remains elusive. We previously showed that histone methyltransferases MLL3/MLL4 and associated PTIP are required for the induction of PPARγ and C/EBPα during adipogenesis. Here, we show MLL3/MLL4/PTIP-associated protein PAGR1 (also known as PA1) cooperates with phosphorylated CREB and ligand-activated glucocorticoid receptor to directly control the induction of C/EBPβ and C/EBPδ in the early phase of adipogenesis. Deletion of Pagr1 in white and brown preadipocytes prevents the induction of C/EBPβ and C/EBPδ and leads to severe defects in adipogenesis. Adipogenesis defects in PAGR1-deficient cells can be rescued by the ectopic expression of C/EBPβ or PPARγ. Finally, the deletion of Pagr1 in Myf5+ precursor cells impairs brown adipose tissue and muscle development. Thus, by controlling the induction of C/EBPβ and C/EBPδ, PAGR1 plays a critical role in adipogenesis.

Farnesoid X Receptor-Mediated Cytoplasmic Translocation of CRTC2 Disrupts CREB-BDNF Signaling in Hippocampal CA1 and Leads to the Development of Depression-Like Behaviors in Mice

Background We recently identified neuronal expression of farnesoid X receptor (FXR), a bile acid receptor known to impair autophagy by inhibiting cyclic adenosine monophosphate response element-binding protein (CREB), a protein whose underfunctioning is linked to neuroplasticity and depression. In this study, we hypothesize that FXR may mediate depression via a CREB-dependent mechanism. Methods Depression was induced in male C57BL6/J mice via chronic unpredictable stress (CUS). Subjects underwent behavioral testing to identify depression-like behaviors. A variety of molecular biology techniques, including viral-mediated gene transfer, Western blot, co-immunoprecipitation, and immunofluorescence, were used to correlate depression-like behaviors with underlying molecular and physiological events. Results Overexpression of FXR, whose levels were upregulated by CUS in hippocampal CA1, induced or aggravated depression-like behaviors in stress-naïve and CUS-exposed mice, while FXR short hairpin RNA (shRNA) ameliorated such symptoms in CUS-exposed mice. The behavioral effects of FXR were found to be associated with changes in CREB-brain-derived neurotrophic factor (BDNF) signaling, as FXR overexpression aggravated CUS-induced reduction in BDNF levels while the use of FXR shRNA or disruption of FXR-CREB signaling reversed the CUS-induced reduction in the phosphorylated CREB and BDNF levels. Molecular analysis revealed that FXR shRNA prevented CUS-induced cytoplasmic translocation of CREB-regulated transcription coactivator 2 (CRTC2); CRTC2 overexpression and CRTC2 shRNA abrogated the regulatory effect of FXR overexpression or FXR shRNA on CUS-induced depression-like behaviors. Conclusions In stress conditions, increased FXR in the CA1 inhibits CREB by targeting CREB and driving the cytoplasmic translocation of CRTC2. Uncoupling of the FXR-CREB complex may be a novel strategy for depression treatment.

Salt-inducible kinase 1 regulates bone anabolism via the CRTC1–CREB–Id1 axis

New bone anabolic agents for the effective treatment of bone metabolic diseases like osteoporosis are of high clinical demand. In the present study, we reveal the function of salt-inducible kinase 1 (SIK1) in regulating osteoblast differentiation. Gene knockdown of SIK1 but not of SIK2 or SIK3 expression in primary preosteoblasts increased osteoblast differentiation and bone matrix mineralization. SIK1 also regulated the proliferation of osteoblastic precursor cells in osteogenesis. This negative control of osteoblasts required the catalytic activity of SIK1. SIK1 phosphorylated CREB regulated transcription coactivator 1 (CRTC1), preventing CRTC1 from enhancing CREB transcriptional activity for the expression of osteogenic genes like Id1. Furthermore, SIK1 knockout (KO) mice had higher bone mass, osteoblast number, and bone formation rate versus littermate wild-type (WT) mice. Preosteoblasts from SIK1 KO mice showed more osteoblastogenic potential than did WT cells, whereas osteoclast generation among KO and WT precursors was indifferent. In addition, bone morphogenic protein 2 (BMP2) suppressed both SIK1 expression as well as SIK1 activity by protein kinase A (PKA)–dependent mechanisms to stimulate osteogenesis. Taken together, our results indicate that SIK1 is a key negative regulator of preosteoblast proliferation and osteoblast differentiation and that the repression of SIK1 is crucial for BMP2 signaling for osteogenesis. Therefore, we propose SIK1 to be a useful therapeutic target for the development of bone anabolic strategies.

Modulation of Phospho-CREB by Systemically Administered Recombinant BDNF in the Hippocampus of the R6/2 Mouse Model of Huntington’s Disease

Huntington’s disease (HD) is an autosomal dominant neurodegenerative disease due to an expansion of a trinucleotide repeats in IT15 gene encoding for the protein huntingtin. Motor dysfunction, cognitive decline, and psychiatric disorder are typical clinical signs of HD. In HD, mutated huntingtin causes a major loss of brain derived neurotrophic factor (BDNF), causing striatal atrophy. Moreover, a key involvement of BDNF was observed in the synaptic plasticity that controls the acquisition and/or consolidation of certain forms of memory. We studied changes in hippocampal BDNF and in CREB in the R6/2 mouse model of HD. Moreover, we investigated if the beneficial effects of systemically administered recombinant BDNF observed in the striatum and cortex had an effect also on the hippocampus. Osmotic minipumps that chronically released recombinant BDNF or saline solution from 4 weeks of age until euthanasia were implanted into R6/2 and wild type mice. Our data show that BDNF is severely decreased in the hippocampus of R6/2 mice, while BDNF treatment restored its physiological levels. Moreover, the chronic administration of recombinant BDNF promoted the increment of phosphorylated CREB protein. Our study demonstrates the involvement of hippocampus in the pathology of R6/2 model of HD and correlates the beneficial effects of BDNF administration with increased hippocampal levels of BDNF and pCREB.