Lumicrine signaling: Extracellular regulation of sperm maturation in the male reproductive tract lumen.

The behaviors of cells, tissues, and organs are controlled by the extracellular environment in addition to their autonomous regulatory system. Dysfunction of extracellular regulatory mechanisms affects not only the development and survival of organisms but also successful reproduction. In this review article, a novel extracellular regulatory mechanism regulating the mammalian male reproductive ability will be briefly summarized. In terrestrial vertebrates, spermatozoa generated in the testis are transported through the lumen of the male reproductive tract and become functionally mature during the transport. Recent studies with gene-modified animals are unveiling the luminal extracellular environment of the reproductive tract to function not only as the pathway of sperm transport and the site of sperm maturation but also as the channel for cellular communication to regulate sperm maturation. Of special interest is the molecular mechanism of lumicrine signaling, a transluminal secreted signal transduction in the male reproductive tract lumen as a master regulator of sperm maturation and male reproductive ability. The general significance of such transluminal signaling in the context of cell biology will also be discussed.

Expression of NELL2/NICOL-ROS1 lumicrine signaling-related molecules in the human male reproductive tract.

The maturation of spermatozoa is a regulated process, influenced by genes expressing essential secreted proteins in the proximal epididymis. Recent genetic studies in rodents have identified the non-sex steroidal molecular signals that regulate gene expression in the proximal epididymis. Germ cells in the testis secrete ligand proteins into the seminiferous tubule lumen The ligand proteins travel through the male reproductive tract lumen to the epididymis, where they bind to receptors, triggering the differentiation of the luminal epithelium for sperm maturation. It is, however, not fully unveiled if such a testis-epididymis trans-luminal signaling mechanism exists in other species, especially humans. In the present study, the rodent-type testis-epididymis trans-luminal signaling in the human male reproductive tract was evaluated in a step-by-step manner by analyzing testis and epididymis gene expression and signaling mediator protein function. There was a significant correlation between the epididymal expressions of mouse genes upregulated by the trans-luminal signaling and those of their human orthologs, as evaluated by the correlation coefficient of 0.604. The transcript expression of NELL2 and NICOL encoding putative ligand proteins was also observed in human testicular cells. In vitro experiments demonstrated that purified recombinant human NELL2 and NICOL formed a molecular complex with similar properties to rodent proteins, which was evaluated by a dissociation equilibrium constant of 110 nM. Recombinant human NELL2 also specifically bound to its putative receptor human ROS1 in vitro. Collectively, these findings suggest that the rodent-type testis-epididymis secreted signaling mechanism is also possible in the human male reproductive tract.

Nell2 regulates the contralateral-versus-ipsilateral visual projection as a domain-specific positional cue.

In mammals with binocular vision, retinal ganglion cell (RGC) axons from each eye project to eye-specific domains in the contralateral and ipsilateral dorsal lateral geniculate nucleus (dLGN), underpinning disparity-based stereopsis. Although domain-specific axon guidance cues that discriminate contralateral and ipsilateral RGC axons have long been postulated as a key mechanism for development of the eye-specific retinogeniculate projection, the molecular nature of such cues has remained elusive. Here, we show that the extracellular glycoprotein Nell2 (neural epidermal growth factor-like-like 2) is expressed in the dorsomedial region of the dLGN, which ipsilateral RGC axons terminate in and contralateral axons avoid. In Nell2 mutant mice, contralateral RGC axons abnormally invaded the ipsilateral domain of the dLGN, and ipsilateral axons terminated in partially fragmented patches, forming a mosaic pattern of contralateral and ipsilateral axon-termination zones. In vitro, Nell2 exerted inhibitory effects on contralateral, but not ipsilateral, RGC axons. These results provide evidence that Nell2 acts as a domain-specific positional label in the dLGN that discriminates contralateral and ipsilateral RGC axons, and that it plays essential roles in the establishment of the eye-specific retinogeniculate projection.

Neural EGFL like 2 expressed in myoepithelial cells and suppressed breast cancer cell migration.

Breast tissue has a branching structure that contains double-layered cells, consisting primarily of luminal epithelial cells inside and myoepithelial cells outside. Ductal carcinoma in situ (DCIS) still has myoepithelial cells surrounding the cancer cells. However, myoepithelial cells disappear in invasive ductal carcinoma. In this study, we detected expression of neural EGFL like (NELL) 2 and one of its receptors, roundabout guidance receptor (ROBO) 3, in myoepithelial and luminal epithelial cells (respectively) in normal breast tissue. NELL2 also was expressed in myoepithelial cells surrounding the non-cancerous intraductal proliferative lesions and DCIS. However, the expression level and proportion of NELL2-positive cells in DCIS were lower than those in normal and non-cancerous intraductal proliferative lesions. ROBO3 expression was decreased in invasive ductal carcinoma compared to that in normal and non-cancerous intraductal proliferative lesions. An evaluation of NELL2's function in breast cancer cell lines demonstrated that full-length NELL2 suppressed cell adhesion and migration in vitro. In contrast, the N-terminal domain of NELL2 increased cell adhesion in the early phase and migration in vitro in some breast cancer cells. These results suggested that full-length NELL2 protein, when expressed in myoepithelial cells, might serve as an inhibitor of breast cancer cell migration.

Expression and regulatory effects on cancer cell behavior of NELL1 and NELL2 in human renal cell carcinoma.

Neural epidermal growth factor-like like (NELL) 1 and 2 constitute a family of multimeric and multimodular extracellular glycoproteins. Although the osteogenic effects of NELL1 and functions of NELL2 in neural development have been reported, their expression and functions in cancer are largely unknown. In this study, we examined expression of NELL1 and NELL2 in renal cell carcinoma (RCC) using clinical specimens and cell lines. We show that, whereas NELL1 and NELL2 proteins are strongly expressed in renal tubules in non-cancerous areas of RCC specimens, their expression is significantly downregulated in cancerous areas. Silencing of NELL1 and NELL2 mRNA expression was also detected in RCC cell lines. Analysis of NELL1/2 promoter methylation status indicated that the CpG islands in the NELL1 and NELL2 genes are hypermethylated in RCC cell lines. NELL1 and NELL2 bind to RCC cells, suggesting that these cells express a receptor for NELL1 and NELL2 that can transduce signals. Furthermore, we found that both NELL1 and NELL2 inhibit RCC cell migration, and NELL1 further inhibits RCC cell adhesion. These results suggest that silencing of NELL gene expression by promoter hypermethylation plays roles in RCC progression by affecting cancer cell behavior.

Haploinsufficiency of ANO6, NELL2 and DBX2 in a boy with intellectual disability and growth delay.

We report on a 10-year-old-boy presenting with moderate intellectual disability (ID), impaired motor skills, hypotonia, growth delay, minor anomalies, misaligned teeth, pectus excavatum, small hands and feet, widely spaced nipples, and a 1.13 Mb de novo deletion on HSA12q12 (chr12:44,830,147-45,964,945 bp, hg19), deleting ANO6, NELL2, and DBX2 and the pseudogenes PLEKHA8P1 and RACGAP1P. We suggest DBX2 and NELL2 as disease-causing genes and their haploinsufficiency to be involved in the psychomotor delay in the patient. DBX2 encodes a homeobox protein, highly expressed during neuronal development and regulating differentiation of interneurons in brain and spinal cord. NELL2 is expressed in most of the central and peripheral nervous system, with highest expression in hippocampus and cerebellum, maximizing during neuronal differentiation. The deletion in our patient is the smallest in HSA12q12 reported to date, and it is included in the deletion carried by four previously reported patients. The clinical presentation of these patients points to the recurrence of the following manifestation, possibly delineating a 12q12 deletion syndrome phenotype: moderate to severe developmental/intellectual delay, hypotonia, postnatal growth retardation, skeletal and dental anomalies, minor facial anomalies including strabismus, down slanting palpebral fissures, and large/low-set ears.

The cytosolic splicing variant of NELL2 inhibits PKCß1 in glial cells.

NELL2 is an abundant glycoprotein containing EGF-like domain in the neural tissues where it has multiple physiological functions by interacting with protein kinase C (PKC). There are two different splicing variant forms of NELL2 identified so far. One is secreted NELL2 (sNELL2) which is a neuron-specific variant and the other is cytosolic NELL2 (cNELL2) which is non-secreted splicing variant of NELL2. Although cNELL2 structure was well characterized, the expression pattern or the cellular function of cNELL2 is not fully determined. In this study, we found that cNELL2 specifically interacts with PKCß isotypes and inhibits PKCß1 through direct binding to the N-terminal pseudosubstrate domain of PKCß1. Here, we also demonstrate that cNELL2 is predominantly expressed and has inhibitory effects on the PKC downstream signaling pathways in astrocytes thereby establishing cNELL2 as an endogenous inhibitor of PKCß1 in glia.

A novel basement membrane-related gene signature for prognosis of lung adenocarcinomas.

BACKGROUND: Lung adenocarcinoma (LUAD) remains a global health concern with its poor prognosis and high mortality. Whether tumor cells invade through the basement membrane (BM) is the key factor to determine the prognosis of LUAD. This study aimed to identify the BM-related gene signatures to improve the overall prognosis of LUAD. MATERIALS & METHODS: A series of bioinformatics analyses were conducted based on TCGA and GEO datasets. Unsupervised consistent cluster analysis was performed, and 500 LUAD patients were assigned to two different groups according to expressions of 222 BM-related genes. The differentially expressed genes (DEGs) between the two clusters were identified, and Lasso regression, ROC curve, univariate and multivariate Cox regression analyses and enrichment analysis were conducted. Besides, ssGSEA, CIBERSORT and ESTIMATE algorithmwere were employed to understand the relationship between the tumor microenvironment (TME) and risk scores. Moreover, single cell clustering and trajectory analyses were performed to further understand the significance of BM-related genes. Finally, qRT-PCR was used to verify the prognosis model. RESULTS: A total of 31 prognostic BM-related genes were determined for LUAD, and a novel 17-mRNA prognostic model named BMsocre was successfully established to predict the overall survival of LUAD patients. The high BMscore group indicated worse prognosis. Seventeen DEGs were enriched mainly in metabolism, ECM-receptor interaction and immune response. In addition, the high-risk group showed higher TMB and lower immune score. The low-risk group had a better immunotherapeutic response where immune escape was less likely. The BMscore model was verified in our patient cohort. Furthermore, NELL2 was mainly expressed in clusters of T cells, and was identified to play a critical role in T-cell differentiation. CONCLUSIONS: A novel BMscore model was successfully established and might be effective for providing guidance to LUAD therapy.

The molecular mechanisms of mammalian sperm maturation regulated by NELL2-ROS1 lumicrine signaling.

In terrestrial vertebrates, spermatozoa generated in the testis are transported through the reproductive tract toward outside the body. In addition to as the pathway of sperm transport, the male reproductive tract also functions as the site of post-testicular sperm maturation and the epididymis, which constitutes the majority of male reproductive tract, and plays central roles in such a sperm maturation. Recent studies with gene-modified animals have been unveiling not only the molecular mechanisms of sperm maturation in the epididymis but also the regulatory system by which the epididymis acquires and executes sperm-maturing functions. In this review, the mechanisms of mammalian sperm maturation will be summarized, based on recent findings, including the lumicrine regulation of sperm maturation.

Neural Epidermal Growth Factor-Like Like Protein 2 Is Expressed in Human Oligodendroglial Cell Types.

Neural epidermal growth factor-like like 2 (NELL2) is a cytoplasmic and secreted glycosylated protein with six epidermal growth factor-like domains. In animal models, NELL2 is predominantly expressed in neural tissues where it regulates neuronal differentiation, polarization, and axon guidance, but little is known about the role of NELL2 in human brain development. In this study, we show that rostral neural stem cells (rNSC) derived from human-induced pluripotent stem cell (hiPSC) exhibit particularly strong NELL2 expression and that NELL2 protein is enriched at the apical side of neural rosettes in hiPSC-derived brain organoids. Following differentiation of human rostral NSC into neurons, NELL2 remains robustly expressed but changes its subcellular localization from >20 small cytoplasmic foci in NSC to one-five large peri-nuclear puncta per neuron. Unexpectedly, we discovered that in human brain organoids, NELL2 is readily detectable in the oligodendroglia and that the number of NELL2 puncta increases as oligodendrocytes mature. Artificial intelligence-based machine learning further predicts a strong association of NELL2 with multiple human white matter diseases, suggesting that NELL2 may possess yet unexplored roles in regulating oligodendrogenesis and/or myelination during human cortical development and maturation.

Transcriptional Regulatory Role of NELL2 in Preproenkephalin Gene Expression.

Preproenkephalin (PPE) is a precursor molecule for multiple endogenous opioid peptides Leu-enkephalin (ENK) and Met-ENK, which are involved in a wide variety of modulatory functions in the nervous system. Despite the functional importance of ENK in the brain, the effect of brain-derived factor(s) on PPE expression is unknown. We report the dual effect of neural epidermal growth factor (EGF)-likelike 2 (NELL2) on PPE gene expression. In cultured NIH3T3 cells, transfection of NELL2 expression vectors induced an inhibition of PPE transcription intracellularly, in parallel with downregulation of protein kinase C signaling pathways and extracellular signal-regulated kinase. Interestingly, these phenomena were reversed when synthetic NELL2 was administered extracellularly. The in vivo disruption of NELL2 synthesis resulted in an increase in PPE mRNA level in the rat brain, suggesting that the inhibitory action of intracellular NELL2 predominates the activation effect of extracellular NELL2 on PPE gene expression in the brain. Biochemical and molecular studies with mutant NELL2 structures further demonstrated the critical role of EGF-like repeat domains in NELL2 for regulation of PPE transcription. These are the first results to reveal the spatio-specific role of NELL2 in the homeostatic regulation of PPE gene expression.

Anoctamin 5 promotes osteosarcoma development by increasing degradation of Nel-like proteins 1 and 2.

Anoctamin 5 (ANO5) is a member of the Anoctamin (ANO) family of calcium-activated chloride channels. Although ANO5 expression is upregulated in various cancers, its role in osteosarcoma remains largely unknown. In this study, bioinformatics analysis, western blot, and immunohistochemical staining revealed that ANO5 was upregulated in osteosarcoma cell lines and osteosarcoma tissues, and ANO5 expression was positively associated with tumor size, tumor grade, and metastasis. Functional experiments demonstrated that inhibition of ANO5 decreased, while ANO5 overexpression increased, osteosarcoma cell proliferation and mobility in vitro. Immunoprecipitation, western blot, and confocal microscopy experiments showed that ANO5 bound to and promoted the degradation of Nel-like proteins 1 (NELL1) and 2 (NELL2). Moreover, a subcutaneous tumor transplantation model revealed that ANO5 knockdown reduced osteosarcoma cell proliferation and increased NELL1 and NELL2 expression in vivo. Finally, rescue experiments showed that knockdown of NELL1 or NELL2 reversed the inhibitory effects of ANO5 knockdown on osteosarcoma cell proliferation and migration. These results demonstrated that upregulation of ANO5 promoted osteosarcoma development by decreasing the stability of the NELL1 and NELL2 proteins and that ANO5 may be an effective target for the treatment of osteosarcoma.

EZH2 suppresses endogenous retroviruses and an interferon response in cancers.

Ewing sarcoma is an aggressive cancer of bone and soft tissue in children. It is characterized by the chromosomal translocation between EWS and an Ets family transcription factor, most commonly FLI1. We recently reported that Ewing sarcoma depends on the autocrine signaling mediated by a cytokine, NELL2. NELL2 signaling stimulates the transcriptional output of EWS-FLI1 through the BAF chromatin remodeling complexes. While studying the impact of NELL2 silencing on Ewing sarcoma, we found that suppression of NELL2 signaling induces the expression of endogenous retroviruses (ERVs) and LINE-1 retrotransposons, an interferon response, and growth arrest. We determined that a histone methyltransferase, EZH2, is the critical downstream target of NELL2 signaling in suppressing ERVs, LINE-1, an interferon response, and growth arrest. We show that EZH2 inhibitors induce ERVs, LINE-1, and an interferon response in a variety of cancer types. These results uncover the role for NELL2-EZH2 signaling in suppressing endogenous virus-like agents and an antiviral response, and suggest the potential utility of EZH2 inhibitors in enhancing anti-tumor immunity.

NELL2-cdc42 signaling regulates BAF complexes and Ewing sarcoma cell growth.

BAF chromatin remodeling complexes play important roles in chromatin regulation and cancer. Here, we report that Ewing sarcoma cells are dependent on the autocrine signaling mediated by NELL2, a secreted glycoprotein that has been characterized as an axon guidance molecule. NELL2 uses Robo3 as the receptor to transmit critical growth signaling. NELL2 signaling inhibits cdc42 and upregulates BAF complexes and EWS-FLI1 transcriptional output. We demonstrate that cdc42 is a negative regulator of BAF complexes, inducing actin polymerization and complex disassembly. Furthermore, we identify NELL2highCD133highEWS-FLI1high and NELL2lowCD133lowEWS-FLI1low populations in Ewing sarcoma, which display phenotypes consistent with high and low NELL2 signaling, respectively. We show that NELL2, CD133, and EWS-FLI1 positively regulate each other and upregulate BAF complexes and cell proliferation in Ewing sarcoma. These results reveal a signaling pathway regulating critical chromatin remodeling complexes and cancer cell proliferation.

The tRNA-Derived Fragment-3017A Promotes Metastasis by Inhibiting NELL2 in Human Gastric Cancer.

tRNA-derived fragments (tRFs) are a new classification of small non-coding RNAs (sncRNAs) derived from the specific cleavage of precursors and mature tRNAs. Accumulating recent evidence has shown that tRFs are frequently abnormal in several cancers. Nevertheless, the role of tRFs in gastric cancer and its mechanism remain unclear. In this study, we found abnormal expression of tRF-3017A (derived from tRNA-Val-TAC) in gastric cancer tissues and cell lines and confirmed its effect on promoting the invasion and migration of gastric cancer cells through functional experiments in vitro. Analysis of clinicopathologic data showed patients with higher tRF-3017A were associated with significantly higher lymph node metastasis. Mechanistic investigation implies that tRF-3017A regulates the tumor suppressor gene NELL2 through forming the RNA-induced silencing complex (RISC) with Argonaute (AGO) proteins. In this study, we found that higher tRF-3017A were associated with significantly higher lymph node metastasis in gastric cancer patients and the tRF-3017A may play a role in promoting the migration and invasion of gastric cancer cells by silencing tumor suppressor NELL2.

GnRH-1 Neural Migration From the Nose to the Brain Is Independent From Slit2, Robo3 and NELL2 Signaling.

Gonadotropin releasing hormone-1 (GnRH-1) neurons play a pivotal role in controlling pubertal onset and fertility once they reach their hypothalamic location. During embryonic development, GnRH-1 neurons migrate from the nasal area to the hypothalamus where they modulate gonadotropin release from the pituitary gland. Defective migration of the GnRH-1 neurons to the brain, lack of GnRH-1 secretion or signaling cause hypogonadotropic hypogonadism (HH), a pathology characterized by delayed or absence of puberty. Binding of the guidance cue Slit2 to the receptor roundabout 3 (Robo3) has been proposed to modulate GnRH-1 cell motility and basal forebrain (bFB) access during migration. However, evidence suggests that Neural EGFL Like 2 (NELL2), not Slit2, binds to Robo3. To resolve this discrepancy, we analyzed GnRH-1 neuronal migration in NELL2, Robo3, and Slit2 knock-out mouse lines. Our data do not confirm a negative effect for monogenic Robo3 and Slit2 mutations on GnRH-1 neuronal migration from the nasal area to the brain. Moreover, we found no changes in GnRH-1 neuronal migration in the brain after NELL2 loss-of-function. However, we found that Slit2 loss-of-function alters the patterning of GnRH-1 cells in the brain, suggesting that Slit2 loss-of-function affects GnRH-1 cell positioning in the brain in a Robo3 independent fashion. Our results challenge previous theories on GnRH-1 neuronal migration mechanisms and provide a new impetus to identify and understand the complex genetic mechanisms causing disorders like Kallmann syndrome (KS) and HH.

Corrigendum: GnRH-1 Neural Migration From the Nose to the Brain Is Independent From Slit2, Robo3 and NELL2 Signaling.

[This corrects the article DOI: 10.3389/fncel.2019.00070.].

A Role of Central NELL2 in the Regulation of Feeding Behavior in Rats.

A brain-enriched secreting signal peptide, NELL2, has been suggested to play multiple roles in the development, survival, and activity of neurons in mammal. We investigated here a possible involvement of central NELL2 in regulating feeding behavior and metabolism. In situ hybridization and an im-munohistochemical approach were used to determine expression of NELL2 as well as its colocalization with proopiomelanocortin (POMC) and neuropeptide Y (NPY) in the rat hypothalamus. To investigate the effect of NELL2 on feeding behavior, 2 nmole of antisense NELL2 oligodeoxynucleotide was administered into the lateral ventricle of adult male rat brains for 6 consecutive days, and changes in daily body weight, food, and water intake were monitored. Metabolic state-dependent NELL2 expression in the hypothalamus was tested in vivo using a fasting model. NELL2 was noticeably expressed in the hypothalamic nuclei controlling feeding behavior. Furthermore, all arcuatic POMC and NPY positive neurons produced NELL2. The NELL2 gene expression in the hypothalamus was up-regulated by fasting. However, NELL2 did not affect POMC and NPY gene expression in the hypothalamus. A blockade of NELL2 production in the hypothalamus led to a reduction in daily food intake, followed by a loss in body weight without a change in daily water intake in normal diet condition. NELL2 did not affect short-term hunger dependent appetite behavior. Our data suggests that hypothalamic NELL2 is associated with appetite behavior, and thus central NELL2 could be a new therapeutic target for obesity.