Characterization of serotonin-5-HTR1E signaling pathways and its role in cell survival.

5-Hydroxytryptamine receptor 1E (5-HTR1E) is reported to activate cyclic AMP (cAMP) and extracellular-signal related kinases (ERK) pathways via its ligands and binding partners, but the detailed mechanism underlying the serotonin-induced 5-HTR1E signaling is still not known. In the present study, we determined the cellular regulators of ERK and cAMP signaling pathways in response to serotonin-induced 5-HTR1E activation in 5-HTR1E overexpressing HEK293 cells. We found that Pertussis Toxin (PTX) treatment completely reversed the effect of serotonin-5-HTR1E mediated signaling on cAMP and ERK pathways, confirming the involvement of a Gαi-linked cascade. We also observed that Gßγ and Gq were not associated with 5-HTR1E activation, while blocking protein kinase A (PKA) inhibited ERK signaling only, and had no effect on cAMP. Additionally, serotonin-stimulated ERK1/2 phosphorylation was similar in 5-HTR1E overexpressing, ß-arrestin-deficient HEK293 cells and is solely dependent on G protein signaling. siRNA mediated gene knockdown studies in SH-SY5Y cells revealed that the inhibition of 5-HTR1E reduced the expression of cMyc, Cyclin D1, Cyclin E and BCL2 genes which are related to cell cycle regulation and survival. MTT assays showed that 5-HTR1E knockdown in SHSY-5Y and U118 cells inhibited cell survival significantly. In addition to the signaling mechanism, we also performed RNA-seq analysis in 5-HTR1E overexpressing HEK293 cells and found that 5-HTR1E can regulate the expression of Receptor activity modifying protein 1 (RAMP1), Nuclear receptor 1 (NR4A1) and other Cyclin genes. These findings indicate that serotonin interaction with 5-HTR1E receptor simultaneously activates cAMP and ERK pathway in HEK293 cells and its expression is important for cell survival.

Hippocampal delivery of neurotrophic factor-α1/carboxypeptidase E gene prevents neurodegeneration, amyloidosis, memory loss in Alzheimer's Disease male mice.

Alzheimer's Disease (AD) is a prevalent neurodegenerative disease characterized by tau hyperphosphorylation, Aß1-42 aggregation and cognitive dysfunction. Therapeutic agents directed at mitigating tau aggregation and clearing Aß1-42, and delivery of growth factor genes (BDNF, FGF2), have ameliorated cognitive deficits, but these approaches did not prevent or stop AD progression. Here we report that viral-(AAV) delivery of Neurotrophic Factor-α1/Carboxypeptidase E (NF-α1/CPE) gene in hippocampus at an early age prevented later development of cognitive deficits as assessed by Morris water maze and novel object recognition assays, neurodegeneration, and tau hyperphosphorylation in male 3xTg-AD mice. Additionally, amyloid precursor protein (APP) expression was reduced to near non-AD levels, and insoluble Aß1-42 was reduced significantly. Pro-survival proteins: mitochondrial Bcl2 and Serpina3g were increased; and mitophagy inhibitor Plin4 and pro-inflammatory protein Card14 were decreased in AAV-NF-α1/CPE treated versus untreated AD mice. Thus NF-α1/CPE gene therapy targets many regulatory components to prevent cognitive deficits in 3xTg-AD mice and has implications as a new therapy to prevent AD progression by promoting cell survival, inhibiting APP overexpression and tau hyperphosphorylation.

The discovery, structure, and function of 5-HTR1E serotonin receptor.

Serotonin (5-hydroxytryptamine, 5-HT) is a unique neurotransmitter which can regulate various biological processes by activating thirteen different receptors. These serotonin receptors are divided into seven different classes based on their structure and functions. Since these receptors co-express in various tissue and cell types and share the same ligand (5-HT), it has been a challenge for the researchers to define specific pathway and separate physiological role for each of these serotonin receptors. Though the evidence of operational diversity of these receptors is continuously emerging, much work remains to be done. 5-HTR1E is a member of 5-HT1 receptor family which belongs to G-protein coupled receptors (GPCRs). Even after three decades since its discovery, 5-HTR1E remains the least explored serotonin receptor. Very high similarity with another family member (5-HTR1F) and its non-existence in mice or rats makes 5-HTR1E a difficult target to study. Despite these challenges, recent findings on the role of 5-HTR1E in neuroprotection and diseases such as cancer, have excited many researchers to explore this receptor in detail. Here, we provide the first review of 5-HTR1E, since its discovery in 1989 to 2023. We highlight the structural and functional characteristics of this important serotonin receptor in detail and propose future directions in developing 5-HTR1E as a drug target. Video Abstract.

Novel interaction between neurotrophic factor-α1/carboxypeptidase E and serotonin receptor, 5-HTR1E, protects human neurons against oxidative/neuroexcitotoxic stress via ß-arrestin/ERK signaling.

Protecting neurons from death during oxidative and neuroexcitotoxic stress is key for preventing cognitive dysfunction. We uncovered a novel neuroprotective mechanism involving interaction between neurotrophic factor-α1 (NF-α1/carboxypeptidase E, CPE) and human 5-HTR1E, a G protein-coupled serotonin receptor with no previously known neurological function. Co-immunoprecipitation and pull-down assays confirmed interaction between NFα1/CPE and 5-HTR1E and 125I NF-α1/CPE-binding studies demonstrated saturable, high-affinity binding to 5-HTR1E in stably transfected HEK293 cells (Kd = 13.82 nM). Treatment of 5-HTR1E stable cells with NF-α1/CPE increased pERK 1/2 and pCREB levels which prevented a decrease in pro-survival protein, BCL2, during H2O2-induced oxidative stress. Cell survival assay in ß-arrestin Knockout HEK293 cells showed that the NF-α1/CPE-5-HTR1E-mediated protection against oxidative stress was ß-arrestin-dependent. Molecular dynamics studies revealed that NF-α1/CPE interacts with 5-HTR1E via 3 salt bridges, stabilized by several hydrogen bonds, independent of the serotonin pocket. Furthermore, after phosphorylating the C-terminal tail and intracellular loop 3 (ICL3) of NF-α1/CPE-5-HTR1E, it recruited ß-arrestin1 by forming numerous salt bridges and hydrogen bonds to ICL2 and ICL3, leading to activation of ß-arrestin1. Immunofluorescence studies showed 5-HTR1E and NF-α1/CPE are highly expressed and co-localized on cell surface of human hippocampal neurons. Importantly, knock-down of 5-HTR1E in human primary neurons diminished the NF-α1/CPE-mediated protection of these neurons against oxidative stress and glutamate neurotoxicity-induced cell death. Thus, NF-α1/CPE uniquely interacts with serotonin receptor 5-HTR1E to activate the ß-arrestin/ERK/CREB/BCL2 pathway to mediate stress-induced neuroprotection.

Exosomal Carboxypeptidase E (CPE) and CPE-shRNA-Loaded Exosomes Regulate Metastatic Phenotype of Tumor Cells.

BACKGROUND: Exosomes promote tumor growth and metastasis through intercellular communication, although the mechanism remains elusive. Carboxypeptidase E (CPE) supports the progression of different cancers, including hepatocellular carcinoma (HCC). Here, we investigated whether CPE is the bioactive cargo within exosomes, and whether it contributes to tumorigenesis, using HCC cell lines as a cancer model. METHODS: Exosomes were isolated from supernatant media of cancer cells, or human sera. mRNA and protein expression were analyzed using PCR and Western blot. Low-metastatic HCC97L cells were incubated with exosomes derived from high-metastatic HCC97H cells. In other experiments, HCC97H cells were incubated with CPE-shRNA-loaded exosomes. Cell proliferation and invasion were assessed using MTT, colony formation, and matrigel invasion assays. RESULTS: Exosomes released from cancer cells contain CPE mRNA and protein. CPE mRNA levels are enriched in exosomes secreted from high- versus low-metastastic cells, across various cancer types. In a pilot study, significantly higher CPE copy numbers were found in serum exosomes from cancer patients compared to healthy subjects. HCC97L cells, treated with exosomes derived from HCC97H cells, displayed enhanced proliferation and invasion; however, exosomes from HCC97H cells pre-treated with CPE-shRNA failed to promote proliferation. When HEK293T exosomes loaded with CPE-shRNA were incubated with HCC97H cells, the expression of CPE, Cyclin D1, a cell-cycle regulatory protein and c-myc, a proto-oncogene, were suppressed, resulting in the diminished proliferation of HCC97H cells. CONCLUSIONS: We identified CPE as an exosomal bioactive molecule driving the growth and invasion of low-metastatic HCC cells. CPE-shRNA loaded exosomes can inhibit malignant tumor cell proliferation via Cyclin D1 and c-MYC suppression. Thus, CPE is a key player in the exosome transmission of tumorigenesis, and the exosome-based delivery of CPE-shRNA offers a potential treatment for tumor progression. Notably, measuring CPE transcript levels in serum exosomes from cancer patients could have potential liquid biopsy applications.

Retraction Note to: Carboxypeptidase E: Elevated Expression Correlated with Tumor Growth and Metastasis in Pheochromocytomas and Other Cancers.

The Editor and the Publisher have retracted this article [1] following an investigation by the National Institutes of Health (NIH) into the primary data used to construct Table 2. This investigation concluded that the data in Table 2 are unreliable.

Cloning, gene regulation, and neuronal proliferation functions of novel N-terminal-truncated carboxypeptidase E/neurotrophic factor-αl variants in embryonic mouse brain.

Carboxypeptidase E (CPE), an exopeptidase involved in proneuropeptide processing, is also a neurotrophic factor, named neurotrophic factor-α1 (NF-α1) and has important roles in neuroprotection, stem cell differentiation, and neurite outgrowth, independent of enzymatic activity. Additionally, an N-terminal-truncated CPE/NF-α1 variant, (CPE/NF-α1)-ΔN, proposed from bioinformatic analysis of GenBank (National Center for Biotechnology Information, Bethesda, MD, USA) DNA sequences and encoding a 40-kDa protein, has been found to be exclusively expressed in embryonic neurons. To investigate the function of (CPE/NF-α1)-ΔN in neurodevelopment, we first cloned (CPE/NF-α1)-ΔN transcripts from an embryonic mouse brain. A rapid amplification of cDNA ends assay, DNA sequencing, and Northern blot revealed 1.9- and 1.73-kb transcripts, which encoded 47- and 40-kDa (CPE/NF-α1)-ΔN proteins, respectively. Those proteins were expressed in embryonic mouse brain. Expression of the 2 (CPE/NF-α1)-ΔN mRNAs surged at embryonic d 10.5, correlating with the time of neurogenesis in the developing brain and also at postnatal d 1. HT22 cells, a mouse hippocampal cell line, transduced with 40 kDa (CPE/NF-α1)-ΔN up-regulated expression of genes involved in embryonic neurodevelopment: insulin-like growth factor binding protein 2 ( IGFBP2), death-associated protein 1, and ephrin A1, which regulate proliferation, programmed cell death, and neuronal migration, respectively. HT22 cells and embryonic cortical neurons overexpressing 40 kDa (CPE/NF-α1)-ΔN exhibited enhanced proliferation, which was inhibited by IGFBP2 short interfering RNA treatment. Thus, 40 kDa (CPE/NF-α1)-ΔN has an important, enzymatically independent role in the regulation of genes critical for neurodevelopment.-Xiao, L., Yang, X., Sharma, V. K., Loh, Y. P. Cloning, gene regulation, and neuronal proliferation functions of novel N-terminal-truncated carboxypeptidase E/neurotrophic factor-αl variants in embryonic mouse brain.

Carboxypeptidase E-∆N Promotes Proliferation and Invasion of Pancreatic Cancer Cells via Upregulation of CXCR2 Gene Expression.

Pancreatic cancer is one of the leading causes of cancer-related mortality worldwide. The molecular basis for the pathogenesis of this disease remains elusive. In this study, we have investigated the role of wild-type Carboxypeptidase E (CPE-WT) and a 40 kDa N-terminal truncated isoform, CPE-ΔN in promoting proliferation and invasion of Panc-1 cells, a pancreatic cancer cell line. Both CPE-WT and CPE-ΔN were expressed in Panc-1 and BXPC-3 pancreatic cancer cells. Immunocytochemical studies revealed that in CPE transfected Panc-1 cells, CPE-ΔN was found primarily in the nucleus, whereas CPE-WT was present exclusively in the cytoplasm as puncta, characteristic of secretory vesicles. Endogenous CPE-WT was secreted into the media. Overexpression of CPE-ΔN in Panc-1 cells resulted in enhancement of proliferation and invasion of these cells, as determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cell proliferation assay and Matrigel invasion assay, respectively. In contrast, the expression of CPE-WT protein at comparable levels to CPE-ΔN in Panc-1 cells resulted in promotion of proliferation but not invasion. Importantly, there was an upregulation of the expression of CXCR2 mRNA and protein in Panc-1 cells overexpressing CPE-ΔN, and these cells exhibited significant increase in proliferation in a CXCR2-dependent manner. Thus, CPE-ΔN may play an important role in promoting pancreatic cancer growth and malignancy through upregulating the expression of the metastasis-related gene, CXCR2.

Neurotrophic Factor-α1: A Key Wnt-ß-Catenin Dependent Anti-Proliferation Factor and ERK-Sox9 Activated Inducer of Embryonic Neural Stem Cell Differentiation to Astrocytes in Neurodevelopment.

Embryonic neurodevelopment involves inhibition of proliferation of multipotent neural stem cells (NSCs) followed by differentiation into neurons, astrocytes and oligodendrocytes to form the brain. We have identified a new neurotrophic factor, NF-α1, which inhibits proliferation and promotes differentiation of NSC/progenitors derived from E13.5 mouse cortex. Inhibition of proliferation of these cells was mediated through negatively regulating the Wnt pathway and decreasing ß-catenin. NF-α1 induced differentiation of NSCs to astrocytes by enhancing Glial Fibrillary Acidic Protein (GFAP) expression through activating the ERK1/2-Sox9 signaling pathway. Cultured E13.5 cortical stem cells from NF-α1-knockout mice showed decreased astrocyte numbers compared to wild-type mice, which was rescued by treatment with NF-α1. In vivo, immunocytochemistry of brain sections and Western blot analysis of neocortex of mice showed a gradual increase of NF-α1 expression from E14.5 to P1 and a surge of GFAP expression at P1, the time of increase in astrogenesis. Importantly, NF-α1-Knockout mice showed ∼49% fewer GFAP positive astrocytes in the neocortex compared to WT mice at P1. Thus, NF-α1 is critical for regulating antiproliferation and cell fate determination, through differentiating embryonic stem cells to GFAP-positive astrocytes for normal neurodevelopment. Stem Cells 2017;35:557-571.

Carboxypeptidase E is a prediction marker for tumor recurrence in early-stage hepatocellular carcinoma.

Tumor recurrence and metastasis are the major causes of death for hepatocellular carcinoma (HCC) patients who are able to receive curative resection. Identifying the predicting biomarkers for tumor recurrence would improve their survival. RNA extracted from fresh frozen tumors and adjacent non-tumor liver tissues of 120 HCC patients were obtained from Taiwan Liver Cancer Network (TLCN) in year 2010 for determination of the carboxypeptidase E (CPE) expression level (including its splicing mutant CPE-ΔN) in the tumor tissue (T) and paired non-tumor liver tissue (N) by real-time quantitative polymerase chain reaction. All patients were male, had chronic hepatitis B virus infection, were in the early pathology stage, and received curative resection. The T/N ratio of the CPE expression level was correlated with the updated survival data from TLCN in 2015. The CPE expression level in the 120 HCC patients was divided into three groups according to the T/N ratio: <1, ≥1 and ≤2, and >2, respectively. By multivariate analyses, the recurrence-free survival (RFS) was only significantly associated with the pathology stage and the CPE expression level. For overall survival (OS), only the CPE expression level was the significant prognostic factor. The CPE expression level was also significantly correlated with the tumor recurrence for both stage I (p = 0.0106) and stage II patients (p = 0.0006). The CPE mRNA expression level in HCC can be a useful biomarker for predicting tumor recurrence in HCC patients who are in the early pathology stage and able to receive curative resection.

Neurotrophic factor-α1 modulates NGF-induced neurite outgrowth through interaction with Wnt-3a and Wnt-5a in PC12 cells and cortical neurons.

Wnt-3a and Wnt-5a signaling activities inhibit and promote neurite outgrowth, respectively, to regulate dendritic and axonal genesis during neurodevelopment. NF-α1, a neurotrophic factor, has been shown to modulate dendritic remodeling and negatively regulate the canonical Wnt-3a pathway. Here, we investigated whether NF-α1 could modify nerve growth factor (NGF)-induced neurite outgrowth through interaction with Wnt-3a and Wnt-5a in PC12 cells and mouse primary cortical neurons. We showed that NGF-induced neurite outgrowth was inhibited by Wnt-3a, and this inhibition was prevented by NF-α1. Western blot analysis revealed that NF-α1 reduced the expression of both ß-catenin in the canonical Wnt-3a pathway and Rho, a downstream effector of Wnt-3a's non-canonical signaling pathway. Treatment of PC12 cells with a ROCK inhibitor prevented the inhibition of NGF-induced neurite outgrowth by Wnt-3a, suggesting that NF-α1 promotes neurite outgrowth in the presence of Wnt-3a by down-regulating its canonical and non-canonical activities. Interestingly, treatment of PC12 cells with Wnt-5a, which formed a complex with NF-α1, induced neurite outgrowth that was enhanced by treatment with the combination of Wnt-5a, NGF, and NF-α1. These effects of NF-α1 on Wnt 3a's and Wnt 5a's regulation of neurite outgrowth in PC12 cells were also demonstrated in primary cultures of mouse cortical neurons. In addition, we showed in PC12 cells that NF-α1 acts by upregulating adenomatous polyposis coli (APC) accumulation at neurite tips, thereby providing positive and negative Wnt-3a/Wnt-5a mediated cues to modulate neurite outgrowth, a process important during neurodevelopment.

A new chromogranin A-dependent angiogenic switch activated by thrombin.

Angiogenesis, the formation of blood vessels from pre-existing vasculature, is regulated by a complex interplay of anti and proangiogenic factors. We found that physiologic levels of circulating chromogranin A (CgA), a protein secreted by the neuroendocrine system, can inhibit angiogenesis in various in vitro and in vivo experimental models. Structure-activity studies showed that a functional anti-angiogenic site is located in the C-terminal region, whereas a latent anti-angiogenic site, activated by cleavage of Q76-K77 bond, is present in the N-terminal domain. Cleavage of CgA by thrombin abrogated its anti-angiogenic activity and generated fragments (lacking the C-terminal region) endowed of potent proangiogenic activity. Hematologic studies showed that biologically relevant levels of forms of full-length CgA and CgA1-76 (anti-angiogenic) and lower levels of fragments lacking the C-terminal region (proangiogenic) are present in circulation in healthy subjects. Blood coagulation caused, in a thrombin-dependent manner, almost complete conversion of CgA into fragments lacking the C-terminal region. These results suggest that the CgA-related circulating polypeptides form a balance of anti and proangiogenic factors tightly regulated by proteolysis. Thrombin-induced alteration of this balance could provide a novel mechanism for triggering angiogenesis in pathophysiologic conditions characterized by prothrombin activation.

A distinct trans-Golgi network subcompartment for sorting of synaptic and granule proteins in neurons and neuroendocrine cells.

Golgi-to-plasma-membrane trafficking of synaptic-like microvesicle (SLMV) proteins, vesicular acetylcholine transporter (VAChT) and synaptophysin (SYN), and a large dense-core vesicle (LDCV) protein, chromogranin A (CgA), was investigated in undifferentiated neuroendocrine PC12 cells. Live cell imaging and 20°C block-release experiments showed that VAChT-GFP, SYN-GFP and CgA-RFP specifically and transiently cohabitated in a distinct sorting compartment during cold block and then separated into synaptic protein transport vesicles (SPTVs) and LDCVs, after release from temperature block. We found that in this trans-Golgi subcompartment there was colocalization of SPTV and LDCV proteins, most significantly with VAMP4 and Golgin97, and to some degree with TGN46, but not at all with TGN38. Moreover, some SNAP25 and VAMP2, two subunits of the exocytic machinery, were also recruited onto this compartment. Thus, in neuroendocrine cells, synaptic vesicle and LDCV proteins converge briefly in a distinct trans-Golgi network subcompartment before sorting into SPTVs and LDCVs, ultimately for delivery to the plasma membrane. This specialized sorting compartment from which SPTVs and LDCVs bud might facilitate the acquisition of common exocytic machinery needed on the membranes of these vesicles.

The novel chromogranin A-derived serpinin and pyroglutaminated serpinin peptides are positive cardiac ß-adrenergic-like inotropes.

Three forms of serpinin peptides, serpinin (Ala26Leu), pyroglutaminated (pGlu)-serpinin (pGlu23Leu), and serpinin-Arg-Arg-Gly (Ala29Gly), are derived from cleavage at pairs of basic residues in the highly conserved C terminus of chromogranin A (CgA). Serpinin induces PN-1 expression in neuroendocrine cells to up-regulate granule biogenesis via a cAMP-protein kinase A-Sp1 pathway, while pGlu-serpinin inhibits cell death. The aim of this study was to test the hypothesis that serpinin peptides are produced in the heart and act as novel ß-adrenergic-like cardiac modulators. We detected serpinin peptides in the rat heart by HPLC and ELISA methods. The peptides included predominantly Ala29Gly and pGlu-serpinin and a small amount of serpinin. Using the Langendorff perfused rat heart to evaluate the hemodynamic changes, we found that serpinin and pGlu-serpinin exert dose-dependent positive inotropic and lusitropic effects at 11-165 nM, within the first 5 min after administration. The pGlu-serpinin-induced contractility is more potent than that of serpinin, starting from 1 nM. Using the isolated rat papillary muscle preparation to measure contractility in terms of tension development and muscle length, we further corroborated the pGlu-serpinin-induced positive inotropism. Ala29Gly was unable to affect myocardial performance. Both pGlu-serpinin and serpinin act through a ß1-adrenergic receptor/adenylate cyclase/cAMP/PKA pathway, indicating that, contrary to the ß-blocking profile of the other CgA-derived cardiosuppressive peptides, vasostatin-1 and catestatin, these two C-terminal peptides act as ß-adrenergic-like agonists. In cardiac tissue extracts, pGlu-serpinin increased intracellular cAMP levels and phosphorylation of phospholamban (PLN)Ser16, ERK1/2, and GSK-3ß. Serpinin and pGlu-serpinin peptides emerge as novel ß-adrenergic inotropic and lusitropic modulators, suggesting that CgA and the other derived cardioactive peptides can play a key role in how the myocardium orchestrates its complex response to sympathochromaffin stimulation.

Trafficking of hormones and trophic factors to secretory and extracellular vesicles: a historical perspective and new hypothesis.

It is well known that peptide hormones and neurotrophic factors are intercellular messengers that are packaged into secretory vesicles in endocrine cells and neurons and released by exocytosis upon the stimulation of the cells in a calcium-dependent manner. These secreted molecules bind to membrane receptors, which then activate signal transduction pathways to mediate various endocrine/trophic functions. Recently, there is evidence that these molecules are also in extracellular vesicles, including small extracellular vesicles (sEVs), which appear to be taken up by recipient cells. This finding raised the hypothesis that they may have functions differentiated from their classical secretory hormone/neurotrophic factor actions. In this article, the historical perspective and updated mechanisms for the sorting and packaging of hormones and neurotrophic factors into secretory vesicles and their transport in these organelles for release at the plasma membrane are reviewed. In contrast, little is known about the packaging of hormones and neurotrophic factors into extracellular vesicles. One proposal is that these molecules could be sorted at the trans-Golgi network, which then buds to form Golgi-derived vesicles that can fuse to endosomes and subsequently form intraluminal vesicles. They are then taken up by multivesicular bodies to form extracellular vesicles, which are subsequently released. Other possible mechanisms for packaging RSP proteins into sEVs are discussed. We highlight some studies in the literature that suggest the dual vesicular pathways for the release of hormones and neurotrophic factors from the cell may have some physiological significance in intercellular communication.

Characterization of serotonin-5-HTR1E signaling pathways and its role in cell survival.

5-Hydroxy tryptamine receptor 1E (5-HTR1E) is reported to activate cAMP and ERK pathways via its ligands and binding partners, but the detailed mechanism underlying the serotonin induced 5-HTR1E signaling is still not known. In the present study, we determined the cellular regulators of ERK and cAMP signaling pathways in response to serotonin induced 5-HTR1E activation in 5-HTR1E overexpressing HEK293 cells. We found that Pertussis Toxin (PTX) treatment completely reversed the effect of serotonin-5-HTR1E mediated signaling on cAMP and ERK pathways, confirming the involvement of a Gαi-linked cascade. We also observed that Gßγ and Gq were not associated with 5-HTR1E activation, while blocking PKA inhibited ERK signaling only, and had no effect on cAMP. Additionally, serotonin-stimulated ERK1/2 phosphorylation was similar in 5-HTR1E overexpressing, ß-arrestin-deficient HEK293 cells and is solely dependent on G protein signaling. siRNA mediated gene knockout studies in SH-SY5Y cells revealed that the inhibition of 5-HTR1E reduced the expression of cMyc, Cyclin D1, Cyclin E and BCL2 genes which are related to cell cycle regulation and survival. MTT assays showed that 5-HTR1E knockdown in SHSY-5Y and U118 cells inhibited cell survival significantly. In addition to the signaling mechanism, we also performed RNA-seq analysis in 5-HTR1E overexpressing HEK293 cells and found that 5-HTR1E can regulate the expression of Receptor activity modifying protein 1 ( RAMP1 ), Nuclear receptor 1 ( NR4A1 ) and other Cyclin genes. These findings indicate that serotonin interaction with 5-HTR1E receptor simultaneously activates cAMP and ERK pathway in HEK293 cells and its expression is important for cell survival.

Carboxypeptidase E conditional knockout mice exhibit learning and memory deficits and neurodegeneration.

Carboxypeptidase E (CPE) is a multifunctional protein with many nonenzymatic functions in various systems. Previous studies using CPE knock-out mice have shown that CPE has neuroprotective effects against stress and is involved in learning and memory. However, the functions of CPE in neurons are still largely unknown. Here we used a Camk2a-Cre system to conditionally knockout CPE in neurons. The wild-type, CPEflox/-, and CPEflox/flox mice were weaned, ear-tagged, and tail clipped for genotyping at 3 weeks old, and they underwent open field, object recognition, Y-maze, and fear conditioning tests at 8 weeks old. The CPEflox/flox mice had normal body weight and glucose metabolism. The behavioral tests showed that CPEflox/flox mice had impaired learning and memory compared with wild-type and CPEflox/- mice. Surprisingly, the subiculum (Sub) region of CPEflox/flox mice was completely degenerated, unlike the CPE full knockout mice, which exhibit CA3 region neurodegeneration. In addition, doublecortin immunostaining suggested that neurogenesis in the dentate gyrus of the hippocampus was significantly reduced in CPEflox/flox mice. Interestingly, TrkB phosphorylation in the hippocampus was downregulated in CPEflox/flox mice, but brain-derived neurotrophic factor levels were not. In both the hippocampus and dorsal medial prefrontal cortex, we observed reduced MAP2 and GFAP expression in CPEflox/flox mice. Taken together, the results of this study demonstrate that specific neuronal CPE knockout leads to central nervous system dysfunction in mice, including learning and memory deficits, hippocampal Sub degeneration and impaired neurogenesis.

Carboxypeptidase E and its splice variants: Key regulators of growth and metastasis in multiple cancer types.

Mechanisms driving tumor growth and metastasis are complex, and involve the recruitment of many genes working in concert with each other. The tumor is characterized by the expression of specific sets of genes depending on its environment. Here we review the role of the carboxypeptidase E (CPE) gene which has been shown to be important in driving growth, survival and metastasis in many cancer types. CPE was first discovered as a prohormone processing enzyme, enriched in endocrine tumors, and later found to be expressed and secreted from many epithelial-derived tumors and cancer cell lines. Numerous studies have shown that besides wild-type CPE, a N-terminal truncated splice variant form of CPE (CPE-ΔN) has been cloned and found to be highly expressed in malignant tumors and cell lines derived from prostate, breast, liver and lung cancers and gliomas. The mechanisms of action of CPE and the splice variant in promoting tumor growth and metastasis in different cancer types are discussed. Mechanistically, secreted CPE activates the Erk/wnt pathways, while CPE-ΔN interacts with HDACs in a protein complex in the nucleus, to recruit various cell cycle genes and metastatic genes, respectively. Clinical studies suggest that CPE and CPE-ΔN mRNA and protein are potential diagnostic and prognostic biomarkers for multiple cancer types, assayed using solid tumors and secreted serum exosomes. CPE has been shown to be a therapeutic target for multiple cancer types. CPE/CPE-ΔN siRNA transported via exosomes and taken up by recipient high metastatic cancer cells, suppressed growth and proliferation of these cells. Thus future studies, delivering CPE/CPE-ΔN siRNA, perhaps via exosomes, to the tumor could be a novel treatment approach to suppress tumor growth and metastasis.