RESUMEN

Background: Apoptosis has been recognized as a critical pathophysiological process during cerebral ischemia. The neuroprotective effect of CART on ischemic brain injury is determined. However, there is little research on the protective effect of CART on neural stem cells (NSCs). Methods: Primary cultured rat NSCs were utilized as the research subject. In vitro oxygen glucose deprivation (OGD) treatment was employed, and NSCs were extracted from SD pregnant rats following previous experimental protocols and identified through cell immunofluorescence staining. The appropriate concentration of CART affecting OGD NSCs was initially screened using Cell Counting Kit-8 (CCK-8) and Lactate Dehydrogenase (LDH) assays. EdU staining and Western blotting (WB) techniques were employed to assess the impact of the suitable CART concentration on the proliferation and apoptosis of OGD NSCs. Finally, Western blot analysis was conducted to investigate the cAMP-response element binding protein (CREB) pathway and expression levels of related proteins after KG-501 treatment in order to elucidate the mechanism underlying apoptosis and proliferation regulation in OGD NSCs. Results: CCK-8 and LDH assays indicated that a concentration of 0.8 nM CART may be the optimal concentration for modulating the proliferation of OGD NSCs. Subsequently, cellular immunofluorescence and EdU detection experiments further confirmed the findings obtained from CCK-8 analysis. Western blot analysis of apoptosis-related protein expression also demonstrated that an appropriate concentration of CART could suppress the apoptosis of OGD NSCs. Finally, Western blotting was conducted to examine the CREB pathway and related protein expression after treatment with KG-501, revealing that an appropriate concentration of CART regulated both apoptosis and proliferation in OGD NSCs through CREB signaling. Conclusion: The concentration of CART at 0.8 nM may be deemed appropriate for inhibiting apoptosis and promoting proliferation in OGD NSCs in vitro. The mechanism maybe through activating the CREB pathway.

RESUMEN

Background: Repetitive transcranial magnetic stimulation (rTMS) is a noninvasive neuromodulation technique that shows promise for the treatment of Parkinson's disease (PD). However, there is still limited understanding of the optimal stimulation frequencies and whether rTMS can alleviate PD symptoms by regulating the CaMKII-CREB-BMAL1 pathway. Methods: A PD mouse model was induced intraperitoneally with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and treated with 1 Hz, 5 Hz, and 10 Hz rTMS. The neurological function, survival of dopaminergic neurons, and protein levels of Tyrosine hydroxylase (TH), α-synuclein(α-syn), and brain-derived neurotrophic factor (BDNF) in the striatum were measured to determine the optimal stimulation frequencies of rTMS treatment in PD mice. The levels of melatonin, cortisol, and the circadian rhythm of Brain and muscle ARNT-like 1 (BMAL1) in PD model mice were detected after optimal frequency rTMS treatment. Additionally, KN-93 and Bmal1siRNA interventions were used to verify that rTMS could alleviate PD symptoms by regulating the CaMKII-CREB-BMAL1 pathway. Results: Administration of 10 Hz rTMS significantly improved neurological function, increased the protein levels of TH and BDNF, and inhibited abnormal aggregation of a-syn. Furthermore, administration of 10 Hz rTMS regulated the secretion profile of cortisol and melatonin and reversed the circadian arrhythmia of BMAL1 expression. After the KN-93 intervention, the MPTP+rTMS+KN-93 group exhibited decreased levels of P- Ca2+/calmodulin-dependent protein kinase II (CaMKII)/CaMKII, P-cAMP-response-element-binding protein (CREB)/CREB, BMALI, and TH. After Bmal1siRNA intervention, the protein levels of BMAL1 and TH were significantly reduced in the MPTP+10 Hz+ Bmal1siRNA group. At the same time, there were no significant changes in the proportions of P-CaMKIIα/CaMKIIα and P-CREB/CREB expression levels. Finally, immunohistochemical analysis showed that the number of TH-positive neurons was high in the MPTP+10 Hz group, but decreased significantly after KN-93 and Bmal1siRNA interventions. Conclusion: Treatment with 10 Hz rTMS alleviated MPTP-induced PD symptoms by regulating the CaMKII-CREB-BMAL1 pathway. This study provides a comprehensive perspective of the therapeutic mechanisms of rTMS in PD.

RESUMEN

Marine thermal fluctuation profoundly influences energy metabolism, physiology, and survival of marine life. In the present study, short-term and long-term high-temperature stresses were found to affect gluconeogenesis by inhibiting PEPCK activity in the Pacific oyster (Crassostrea gigas), which is a globally distributed species that encounters significant marine thermal fluctuations in intertidal zones worldwide. CgCREBL2, a key molecule in the regulation of gluconeogenesis, plays a critical role in the transcriptional regulation of PEPCK in gluconeogenesis against high-temperature stress. CgCREBL2 was able to increase the transcription of CgPEPCK by either binding the promoter of CgPEPCK gene or activating CgPGC-1α and CgHNF-4α after short-term (6 h) high-temperature stress, while only by binding CgPEPCK after long-term (60 h) high-temperature stress. These findings will further our understanding of the effect of marine thermal fluctuation on energy metabolism on marine organisms.

Asunto(s)

RESUMEN

BACKGROUND AND AIMS: In vivo induction of alcoholic chronic pancreatitis (ACP) causes significant acinar damage, increased fibroinflammatory response, and heightened activation of cyclic response element binding protein 1 (CREB) when compared with alcohol (A) or chronic pancreatitis (CP) mediated pancreatic damage. However, the study elucidating the cooperative interaction between CREB and the oncogenic Kras G12D/+ (Kras*) in promoting pancreatic cancer progression with ACP remains unexplored. METHODS: Experimental ACP induction was established in multiple mouse models, followed by euthanization of the animals at various time intervals during the recovery periods. Tumor latency was determined in these mice cohorts. Here, we established CREB deletion (Creb fl/fl ) in Ptf1a CreERTM/+ ;LSL-Kras G12D+/-(KC) genetic mouse models (KCC-/-). Western blot, phosphokinase array, and qPCR were used to analyze the pancreata of Ptf1a CreERTM+/-, KC and KCC -/- mice. The pancreata of ACP-induced KC mice were subjected to single-cell RNA sequencing (scRNAseq). Further studies involved conducting lineage tracing and acinar cell explant cultures. RESULTS: ACP induction in KC mice had detrimental effects on the pancreatic damage repair mechanism. The persistent existence of acinar cell-derived ductal lesions demonstrated a prolonged state of hyperactivated CREB. Persistent CREB activation leads to acinar cell reprogramming and increased pro-fibrotic inflammation in KC mice. Acinar-specific Creb ablation reduced advanced PanINs lesions, hindered tumor progression, and restored acinar cell function in ACP-induced mouse models. CONCLUSIONS: Our findings demonstrate that CREB cooperates with Kras* to perpetuate an irreversible ADM and PanIN formation. Moreover, CREB sustains oncogenic activity to promote the progression of premalignant lesions toward cancer in the presence of ACP.

RESUMEN

Obesity is a multifactorial disease that is part of today's epidemic and also increases the risk of other metabolic diseases. Long noncoding RNAs (lncRNAs) provide one tier of regulatory mechanisms to maintain metabolic homeostasis. Although lncRNAs are a significant constituent of the mammalian genome, studies aimed at their metabolic significance, including obesity, are only beginning to be addressed. Here, a developmentally regulated lncRNA, termed as obesity related (Obr), whose expression in metabolically relevant tissues such as skeletal muscle, liver, and pancreas is altered in diet-induced obesity, is identified. The Clone 9 cell line and high-fat diet-induced obese Wistar rats are used as a model system to verify the function of Obr. By using stable expression and antisense oligonucleotide-mediated downregulation of the expression of Obr followed by different molecular biology experiments, its role in lipid metabolism is verified. It is shown that Obr associates with the cAMP response element-binding protein (Creb) and activates different transcription factors involved in lipid metabolism. Its association with the Creb histone acetyltransferase complex, which includes the cAMP response element-binding protein (CBP) and p300, positively regulates the transcription of genes involved in lipid metabolism. In addition, Obr is regulated by Pparγ in response to lipid accumulation.

Asunto(s)

RESUMEN

Monitoring inflammatory cytokines is crucial for assessing healing process and photobiomodulation (PBM) enhances wound healing. Meanwhile, cAMP response element-binding protein (CREB) is a regulator of cellular metabolism and proliferation. This study explored potential links between inflammatory cytokines and the activity of CREB in PBM-treated wounds. A total of 48 seven-week-old male SD rats were divided into four groups (wound location, skin or oral; treatment method, natural healing or PBM treatment). Wounds with a 6 mm diameter round shape were treated five times with an 808 nm laser every other day (total 60 J). The wound area was measured with a caliper and calculated using the elliptical formula. Histological analysis assessed the epidermal regeneration and collagen expression of skin and oral tissue with H&E and Masson's trichrome staining. Pro-inflammatory (TNF-α) and anti-inflammatory (TGF-ß) cytokines were quantified by RT-PCR. The ratio of phosphorylated CREB (p-CREB) to unphosphorylated CREB was identified through Western blot. PBM treatment significantly reduced the size of the wounds on day 3 and day 7, particularly in the skin wound group (p < 0.05 on day 3, p < 0.001 on day 7). The density of collagen expression was significantly higher in the PBM treatment group (in skin wound, p < 0.05 on day 3, p < 0.001 on day 7, and p < 0.05 on day 14; in oral wound, p < 0.01 on day 7). The TGF-ß/TNF-α ratio and the p-CREB/CREB ratio showed a parallel trend during wound healing. Our findings suggested that the CREB has potential as a meaningful marker to track the wound healing process.

Asunto(s)

RESUMEN

Particulate matter (PM), released into the air by a variety of natural and human activities, is a key indicator of air pollution. Although PM is known as the extensive health hazard to affect a variety of illness, few studies have specifically investigated the effects of PM10 exposure on schizophrenic development. In the present study, we aimed to investigate the impact of PM10 on MK-801, N-methyl-D-aspartate (NMDA) receptor antagonist, induced schizophrenia-like behaviors in C57BL/6 mouse. Preadolescent mice were exposed PM10 to 3.2 mg/m3 concentration for 4 h/day for 2 weeks through a compartmentalized whole-body inhalation chamber. After PM10 exposure, we conducted behavioral tests during adolescence and adulthood to investigate longitudinal development of schizophrenia. We found that PM10 exacerbated schizophrenia-like behavior, such as psychomotor agitation, social interaction deficits and cognitive deficits at adulthood in MK-801-induced schizophrenia animal model. Furthermore, the reduced expression levels of brain-derived neurotrophic factor (BDNF) and the phosphorylation of BDNF related signaling molecules, extracellular signal-regulated kinase (ERK) and cAMP response element-binding protein (CREB), were exacerbated by PM10 exposure in the adult hippocampus of MK-801-treated mice. Thus, our present study demonstrates that exposure to PM10 in preadolescence exacerbates the cognitive impairment in animal model of schizophrenia, which are considered to be facilitated by the decreased level of BDNF through reduced ERK-CREB expression.

Asunto(s)

RESUMEN

Colorful shells in mollusks are commonly attributable to the presence of biological pigments. In Pacific oysters, the inheritance patterns of several shell colors have been investigated, but little is known about the molecular mechanisms of melanogenesis and pigmentation. cAMP-response element binding proteins (CREB) are important transcription factors in the cAMP-mediated melanogenesis pathway. In this study, we characterized two CREB genes (CREB3L2 and CREB3L3) from Pacific oysters. Both of them contained a conserved DNA-binding and dimerization domain (a basic-leucine zipper domain). CREB3L2 and CREB3L3 were expressed highly in the mantle tissues and exhibited higher expression levels in the black-shell oyster than in the white. Masson-Fontana melanin staining and immunofluorescence analysis showed that the location of CREB3L2 protein was generally consistent with the distribution of melanin in oyster edge mantle. Dual-luciferase reporter assays revealed that CREB3L2 and CREB3L3 could activate the microphthalmia-associated transcription factor (MITF) promoter and this process was regulated by the level of cAMP. Additionally, we found that cAMP regulated melanogenic gene expression through the CREB-MITF-TYR axis. These results implied that CREB3L2 and CREB3L3 play important roles in melanin synthesis and pigmentation in Pacific oysters.

Asunto(s)

RESUMEN

Melatonin is a molecule ubiquitous in nature and involved in several physiological functions. In the brain, melatonin is converted to N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK) and then to N1-acetyl-5-methoxykynuramine (AMK), which has been reported to strongly enhance long-term object memory formation. However, the synthesis of AMK in brain tissues and the underlying mechanisms regarding memory formation remain largely unknown. In the present study, young and old individuals from a melatonin-producing strain, C3H/He mice, were employed. The amount of AMK in the pineal gland and plasma was very low compared with those of melatonin at night; conversely, in the hippocampus, the amount of AMK was higher than that of melatonin. Indoleamine 2, 3-dioxygenase (Ido) mRNA was expressed in multiple brain tissues, whereas tryptophan 2,3-dioxygenase (Tdo) mRNA was expressed only in the hippocampus, and its lysate had melatonin to AFMK conversion activity, which was blocked by the TDO inhibitor. The expression levels of phosphorylated cAMP response element binding protein (CREB) and PSD-95 in whole hippocampal tissue were significantly increased with AMK treatment. Before increasing in the whole tissue, CREB phosphorylation was significantly enhanced in the nuclear fraction. In the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, we found that downregulated genes in hippocampus of old C3H/He mice were more enriched for long-term potentiation (LTP) pathway. Gene set enrichment analysis showed that LTP and neuroactive receptor interaction gene sets were enriched in hippocampus of old mice. In addition, Ido1 and Tdo mRNA expression was significantly decreased in the hippocampus of old mice compared with young mice, and the decrease in Tdo mRNA was more pronounced than Ido1. Furthermore, there was a higher decrease in AMK levels, which was less than 1/10 that of young mice, than in melatonin levels in the hippocampus of old mice. In conclusion, we first demonstrated the Tdo-related melatonin to AMK metabolism in the hippocampus and suggest a novel mechanism of AMK involved in LTP and memory formation. These results support AMK as a potential therapeutic agent to prevent memory decline.

Asunto(s)

RESUMEN

Methamphetamine (METH) can potentially disrupt neurotransmitters activities in the central nervous system (CNS) and cause neurotoxicity through various pathways. These pathways include increased production of reactive nitrogen and oxygen species, hypothermia, and induction of mitochondrial apoptosis. In this study, we investigated the long-term effects of METH addiction on the structural changes in the amygdala of postmortem human brains and the involvement of the brain- cAMP response element-binding protein/brain-derived neurotrophic factor (CREB/BDNF) and Akt-1/GSK3 signaling pathways. We examined ten male postmortem brains, comparing control subjects with chronic METH users, using immunohistochemistry, real-time polymerase chain reaction (to measure levels of CREB, BDNF, Akt-1, GSK3, and tumor necrosis factor-α [TNF-α]), Tunnel assay, stereology, and assays for reactive oxygen species (ROS), glutathione disulfide (GSSG), and glutathione peroxidase (GPX). The findings revealed that METH significantly reduced the expression of BDNF, CREB, Akt-1, and GPX while increasing the levels of GSSG, ROS, RIPK3, GSK3, and TNF-α. Furthermore, METH-induced inflammation and neurodegeneration in the amygdala, with ROS production mediated by the CREB/BDNF and Akt-1/GSK3 signaling pathways.

RESUMEN

For many decades, our understanding of G protein-coupled receptor (GPCR) activity and cyclic AMP (cAMP) signaling was limited exclusively to the plasma membrane. However, a growing body of evidence has challenged this view by introducing the concept of endocytosis-dependent GPCR signaling. This emerging paradigm emphasizes not only the sustained production of cAMP but also its precise subcellular localization, thus transforming our understanding of the spatiotemporal organization of this process. Starting from this alternative point of view, our recent work sheds light on the role of an endocytosis-dependent calcium release from the endoplasmic reticulum in the control of nuclear cAMP levels. This is achieved through the activation of local soluble adenylyl cyclase, which in turn regulates the activation of local protein kinase A (PKA) and downstream transcriptional events. In this review, we explore the dynamic evolution of research on cyclic AMP signaling, including the findings that led us to formulate the novel three-wave hypothesis. We delve into how we abandoned the paradigm of cAMP generation limited to the plasma membrane and the changing perspectives on the rate-limiting step in nuclear PKA activation.

Asunto(s)

RESUMEN

OBJECTIVE: The main purpose of the present study was to assess the beneficial effect of Lactobacillus plantarum GM11 (LacP GM11), screened from Sichuan traditional fermented food, in depressive rats induced by chronic unpredictable mild stress (CUMS). METHODS: Male SPF SD rats were randomly assigned to 3 groups: the control group, CUMS group and CUMS + LacP GM11 group (n = 10). The rats in the CUMS and LacP GM11 groups received CUMS stimulation for 42 d. The behavioral tests and levels of monoamine neurotransmitter, glucocorticoid hormone and brain-derived neurotrophic factor (BDNF) in the serum and hippocampus were measured. The effects of LacP GM11 on the mRNA and protein expression of BDNF and cAMP response element binding protein (CREB) in the hippocampus were also investigated. RESULTS: After supplementation for 21 d, LacP GM11 was associated with alleviation of depressive-like behavior, not anxiety-like behavior, in depressive rats. LacP GM11 increased the levels of 5-hydroxytryptamine (5-HT) and BDNF and decreased the level of cortisol (CORT) in the serum and hippocampus in depressed rats. In addition, treatment with LacP GM11 also increased the mRNA and protein expression of BDNF and CREB in the hippocampus. CONCLUSIONS: This work has revealed that LacP GM11 has potential beneficial effects on depression. This effect might be related to alleviating monoamine neurotransmitter deficiency, HPA axis hyperfunction and CREB-BDNF signaling pathway downregulation. This study demonstrates that LacP GM11 could be a potential therapeutic approach to treat depression and other mental health problems.

Asunto(s)

RESUMEN

Objective To investigate the ameliorating effect of salidroside(SAL)on cisplatin(CIS)-induced damages of cochlear hair cells(CHC)and spiral ganglion neurons(SGNs)and its relationship with cyclic adenosine monophosphate(cAMP)/protein kinase A(PKA)/cAMP response element binding protein(CREB)pathway.Methods The cochlear basilar membranes of newborn C 57BL/6 mice were isolated and separated into control(C)group,CIS group,SAL group,SAL+SQ22536(cAMP inhibitor)group and SAL+H-89(PKA inhibitor)group,20 per group.Immunofluorescence staining was applied to observe the damages of CHC and SGNs.The kits were applied to detect the contents of ROS and cAMP in the basement membrane of the cochlea.Western blot was applied to detect the protein levels of PKA,p-CREB,CREB,Bcl-2,BDNF,and NF-M.Results CHC in CIS group were disorderly arranged and enlarged in size,SGNs had fragmented nuclei and lost neurites.SAL alleviated the damages of CHC and SGNs.Compared with the C group,the numbers of CHC and SGNs in the CIS group were less(P＜0.05),the contents of ROS and cAMP,and the levels of PKA,BDNF,NF-M,Bcl-2 proteins and p-CREB/CREB were higher(P＜0.05).Compared with the CIS group,the numbers of CHC and SGNs in the SAL group were higher(P＜0.05),the content of ROS was lower(P＜0.05),the content of cAMP,and the levels of PKA,BD-NF,NF-M,Bcl-2 proteins and p-CREB/CREB were higher(P＜0.05).Both SQ22536 and H-89 reversed the pro-tective effects of SAL on CHC and SGNs.Conclusion SAL may promote the expression of anti-apoptotic proteins and neuroprotective factors by activating the cAMP/PKA/CREB pathway to alleviate the damages of CHC and SGNs caused by CIS.

RESUMEN

AIM:To observe the effect of acupuncture on adenosine A1 receptor(A1R)in the caudate puta-men(CPu)of complete Freund's adjuvant(CFA)rats,and to explore the potential mechanism of acupuncture in treat-ment of inflammatory pain.METHODS:Sixty-four 6～8-week-old male Wistar rats were randomly divided into saline group,model group(CFA group),CFA+manual acupuncture(MA)group,CFA+solvent dimethyl sulfoxide(DMSO)group,CFA+A1R agonist 2-chloro-N6-cyclopentyladenosine(CCPA)group,CFA+A1R antagonist 8-cyclopentyl-1,3-di-propylxanthine(DPCPX)group,CFA+MA+DMSO group and CFA+MA+DPCPX group.In MA groups,on the 2nd day af-ter modeling,the rats were needled at Zusanli points on both sides,30 min at a time,once per day,for 7 d.Pain threshold of plantar thermal radiation was used to observe the pain response of the rats.The content of cyclic adenosine monophos-phate(cAMP)in the CPu was detected by ELISA.The protein expression and phosphorylation levels of protein kinase A(PKA)and cAMP response element-binding protein(CREB)were detected by Western blot.The expression of A1R in the CPu was detected by immunofluorescence staining.RESULTS:Compared with saline group,CFA modeling signifi-cantly lowered the thermal pain threshold of the rats(P<0.01).Compared with CFA group,the thermal pain threshold of the rats in CFA+MA group and CFA+CCPA group was significantly increased(P<0.05 or P<0.01).Compared with CFA+ MA+DMSO group,the thermal pain threshold of the rats in CFA+MA+DPCPX group was decreased(P<0.05).Compared with CFA group,A1R protein relative expression level and positive cells in the CPu of the rats in CFA+MA group were in-creased(P<0.05 or P<0.01).Compared with saline group,cAMP content and p-CREB protein level in the CPu of the rats in CFA+MA group were decreased(P<0.05).Compared with CFA+DMSO group,cAMP content and p-CREB pro-tein level in CFA+MA+DMSO and CFA+CCPA groups were significantly decreased(P<0.01).Compared with CFA+MA+ DMSO group,the levels of cAMP,p-PKA and p-CREB in CFA+MA+DPCPX group were significantly increased(P<0.05 or P<0.01).CONCLUSION:Acupuncture on bilateral Zusanli can relieve inflammatory pain in CFA rats,and its mech-anism may be related to A1R/cAMP/p-CREB signaling pathway.

RESUMEN

ObjectiveTo decipher the mechanism of Wenxiao powder in alleviating corticosterone-induced depression-like behaviors in mice. MethodMale ICR mice were randomized into normal， model， paroxetine （20 mg·kg-1）， and low- and high-dose （3.27， 6.54 g·kg-1， respectively） Wenxiao powder groups. The mice in normal and model groups received equal volume of saline. Other groups except the normal group were injected with corticosterone subcutaneously 0.5 h after gavage to induce depression. Mice were tested for depression-like behaviors after drug administration. Enzyme-linked immunosorbent assay （ELISA） was performed to measure the corticosterone content in the serum. Nissl staining was performed to observe the damage of hippocampal neurons. Immunofluorescence staining was employed to observe the expression of double cortin （DCX） in the dentate gyrus （DG） of the hippocampus. Western blot was employed to determine the expression of proteins in the brain-derived neurotrophic factor （BDNF）/tyrosine kinase receptor B （TrkB）/extracellular signal-regulated kinase （ERK）/cAMP-response element-binding protein （CREB） pathway in the hippocampus. ResultCompared with the normal group， the model group showed decreased sucrose preference rate， increased immobility time in the tail suspension test （P<0.01）， and reduced residence time in the central area of the open field and the total movement distance （P<0.05， P<0.01）. In addition， the modeling elevated the corticosterone level in the serum （P<0.01）， decreased the volume and intensified the nuclear staining of hippocampal neurons in the DG area， reduced the expression of DCX in the DG area， and down-regulated the protein levels of BDNF， phosphorylated （p）-TrkB， p-ERK， and p-CREB in the hippocampus （P<0.05， P<0.01）. Compared with the model group， low-dose Wenxiao powder improved the mouse behavivors in the sucrose preference， open field， and tail suspension tests （P<0.05， P<0.01）， and high-dose Wenxiao powder improved the behaviors in the sucrose preference and open field tests （P<0.05， P<0.01）. In addition， Wenxiao powder lowered the serum corticosterone level （P<0.01） and recovered the structure and morphology of neurons with obvious nuclei and presence of Nissl bodies in the DG area of the hippocampus. Moreover， Wenxiao powder at both doses promoted the expression of DCX in the DG area， and high-dose Wenxiao powder up-regulated the protein levels of BDNF， p-TrkB， p-ERK， and p-CREB in the hippocampus （P<0.05， P<0.01）. ConclusionWenxiao powder can alleviate corticosterone-induced depression-like behaviors and promote neurogenesis in mice possibly by activating the BDNF/TrkB/ERK/CREB signaling pathway.

RESUMEN

High-level expression of recombinant proteins in mammalian cells has long been an area of interest. Inefficient transcription machinery is often an obstacle in achieving high-level expression of recombinant proteins in mammalian cells. Synthetic promoters have been developed to improve the transcription efficiency, but have achieved limited success due to the limited availability of transcription factors (TFs). Here, we present a TF-engineering approach to mitigate the transcriptional bottlenecks of recombinant proteins. This includes: (i) identification of cAMP response element binding protein (CREB) as a candidate TF by searching for TFs enriched in the cytomegalovirus (CMV) promoter-driven high-producing recombinant Chinese hamster ovary (rCHO) cell lines via transcriptome analysis, (ii) confirmation of transcriptional limitation of active CREB in rCHO cell lines, and (iii) direct activation of the transgene promoter by expressing constitutively active CREB at non-cytotoxic levels in rCHO cell lines. With the expression of constitutively active VP16-CREB, the production of therapeutic proteins, such as monoclonal antibody and etanercept, in CMV promoter-driven rCHO cell lines was increased up to 3.9-fold. VP16-CREB was also used successfully with synthetic promoters containing cAMP response elements. Taken together, this strategy to introduce constitutively active TFs into cells is a useful means of overcoming the transcriptional limitations in recombinant mammalian cells.

Asunto(s)

RESUMEN

BACKGROUND: New therapies are urgently needed in melanoma, particularly in late-stage patients not responsive to immunotherapies and kinase inhibitors. To uncover novel potentiators of T cell anti-tumor immunity, we carried out an ex vivo pharmacological screen and identified 5-Nonyloxytryptamine (5-NL), a serotonin agonist, as increasing the ability of T cells to target tumor cells. METHODS: The pharmacological screen utilized lymphocytic choriomeningitis virus (LCMV)-primed splenic T cells and melanoma B16.F10 cells expressing the LCMV gp33 CTL epitope. In vivo tumor growth in C57BL/6 J and NSG mice, in vivo antibody depletion, flow cytometry, immunoblot, CRISPR/Cas9 knockout, histological and RNA-Seq analyses were used to decipher 5-NL's immunomodulatory effects in vitro and in vivo. RESULTS: 5-NL delayed tumor growth in vivo and the phenotype was dependent on the hosts' immune system, specifically CD8+ T cells. 5-NL's pro-immune effects were not directly consequential to T cells. Rather, 5-NL upregulated antigen presenting machinery in melanoma and other tumor cells in vitro and in vivo without increasing PD-L1 expression. Mechanistic studies indicated that 5-NL's induced MHC-I expression was inhibited by pharmacologically preventing cAMP Response Element-Binding Protein (CREB) phosphorylation. Importantly, 5-NL combined with anti-PD1 therapy showed significant improvement when compared to single anti-PD-1 treatment. CONCLUSIONS: This study demonstrates novel therapeutic opportunities for augmenting immune responses in poorly immunogenic tumors.

Asunto(s)

RESUMEN

Huntington's disease (HD) is a disorder caused by an abnormal expansion of trinucleotide CAG repeats within the huntingtin (Htt) gene. Under normal conditions, the CREB Binding Protein interacts with CREB elements and acetylates Lysine 27 of Histone 3 to direct the expression of several genes. However, mutant Htt causes depletion of CBP, which in turn induces altered histone acetylation patterns and transcriptional deregulation. Here, we have studied a differential expression analysis and H3K27ac variation in 4- and 6-week-old R6/2 mice as a model of juvenile HD. The analysis of differential gene expression and acetylation levels were integrated into Gene Regulatory Networks revealing key regulators involved in the altered transcription cascade. Our results show changes in acetylation and gene expression levels that are related to impaired neuronal development, and key regulators clearly defined in 6-week-old mice are proposed to drive the downstream regulatory cascade in HD. Here, we describe the first approach to determine the relationship among epigenetic changes in the early stages of HD. We determined the existence of changes in pre-symptomatic stages of HD as a starting point for early onset indicators of the progression of this disease.

Asunto(s)

RESUMEN

Objective: To investigate the regulatory effects of miR-26a-5p on the osteogenic differentiation of adipose-derived mesenchymal stem cells (ADSCs) by regulating cAMP response element binding protein 1 (CREB1). Methods: The adipose tissues of four 3-4 weeks old female C57BL/6 mice were collected and the cells were isolated and cultured by digestion separation method. After morphological observation and identification by flow cytometry, the 3rd-generation cells were subjected to osteogenic differentiation induction. At 0, 3, 7, and 14 days after osteogenic differentiation induction, the calcium deposition was observed by alizarin red staining, ALP activity was detected, miR- 26a-5p and CREB1 mRNA expressions were examined by real-time fluorescence quantitative PCR, and CREB1 protein and its phosphorylation (phospho-CREB1, p-CREB1) level were measured by Western blot. After the binding sites between miR-26a-5p and CREB1 was predicted by the starBase database, HEK-293T cells were used to conduct a dual-luciferase reporter gene experiment to verify the targeting relationship (represented as luciferase activity after 48 hours of culture). Finally, miR-26a-p inhibitor (experimental group) and the corresponding negative control (control group) were transfected into ADSCs. Alizarin red staining, ALP activity, real-time fluorescent quantitative PCR (miR-26a-5p) and Western blot [CREB1, p-CREB1, Runt-related transcription factor 2 (RUNX2), and osteocalcin (OCN)] were performed at 7 and 14 days after osteogenic induction culture. Results: The cultured cells were identified as ADSCs. With the prolongation of osteogenic induction culture, the number of calcified nodules and ALP activity significantly increased ( P<0.05). The relative expression of miR-26a-5p in the cells gradually decreased, while the relative expressions of CREB1 mRNA and protein, as well as the relative expression of p-CREB1 protein were increased. The differences were significant between 7, 14 days and 0 day ( P<0.05). There was no significant difference in p-CREB1/CREB1 between different time points ( P>0.05). The starBase database predicted that miR-26a-5p and CREB1 had targeted binding sequences, and the dual-luciferase reporter gene experiment revealed that overexpression of miR-26a-5p significantly suppressed CREB1 wild-type luciferase activity ( P<0.05). After 7 and 14 days of osteogenic induction, compared with the control group, the number of calcified nodules, ALP activity, and relative expressions of CREB1, p-CREB1, OCN, and RUNX2 proteins in the experimental group significantly increased ( P<0.05). There was no significant difference in p-CREB1/CREB1 between the two groups ( P>0.05). Conclusion: Knocking down miR-26a-5p promoted the osteogenic differentiation of ADSCs by up-regulating CREB1 and its phosphorylation.

Asunto(s)

RESUMEN

Deregulated inflammation is a critical feature driving the progression of tumors harboring mutations in the liver kinase B1 (LKB1), yet the mechanisms linking LKB1 mutations to deregulated inflammation remain undefined. Here, we identify deregulated signaling by CREB-regulated transcription coactivator 2 (CRTC2) as an epigenetic driver of inflammatory potential downstream of LKB1 loss. We demonstrate that LKB1 mutations sensitize both transformed and non-transformed cells to diverse inflammatory stimuli, promoting heightened cytokine and chemokine production. LKB1 loss triggers elevated CRTC2-CREB signaling downstream of the salt-inducible kinases (SIKs), increasing inflammatory gene expression in LKB1-deficient cells. Mechanistically, CRTC2 cooperates with the histone acetyltransferases CBP/p300 to deposit histone acetylation marks associated with active transcription (i.e., H3K27ac) at inflammatory gene loci, promoting cytokine expression. Together, our data reveal a previously undefined anti-inflammatory program, regulated by LKB1 and reinforced through CRTC2-dependent histone modification signaling, that links metabolic and epigenetic states to cell-intrinsic inflammatory potential.

Asunto(s)