Nuclear High Mobility Group A2 (HMGA2) Interactome Revealed by Biotin Proximity Labeling.

The non-histone chromatin binding protein High Mobility Group AT-hook protein 2 (HMGA2) has important functions in chromatin remodeling, and genome maintenance and protection. Expression of HMGA2 is highest in embryonic stem cells, declines during cell differentiation and cell aging, but it is re-expressed in some cancers, where high HMGA2 expression frequently coincides with a poor prognosis. The nuclear functions of HMGA2 cannot be explained by binding to chromatin alone but involve complex interactions with other proteins that are incompletely understood. The present study used biotin proximity labeling, followed by proteomic analysis, to identify the nuclear interaction partners of HMGA2. We tested two different biotin ligase HMGA2 constructs (BioID2 and miniTurbo) with similar results, and identified known and new HMGA2 interaction partners, with functionalities mainly in chromatin biology. These HMGA2 biotin ligase fusion constructs offer exciting new possibilities for interactome discovery research, enabling the monitoring of nuclear HMGA2 interactomes during drug treatments.

Ubiquitin Proteasome Gene Signatures in Ependymoma Molecular Subtypes.

The ubiquitin proteasome system (UPS) is critically important for cellular homeostasis and affects virtually all key functions in normal and neoplastic cells. Currently, a comprehensive review of the role of the UPS in ependymoma (EPN) brain tumors is lacking but may provide valuable new information on cellular networks specific to different EPN subtypes and reveal future therapeutic targets. We have reviewed publicly available EPN gene transcription datasets encoding components of the UPS pathway. Reactome analysis of these data revealed genes and pathways that were able to distinguish different EPN subtypes with high significance. We identified differential transcription of several genes encoding ubiquitin E2 conjugases associated with EPN subtypes. The expression of the E2 conjugase genes UBE2C, UBE2S, and UBE2I was elevated in the ST_EPN_RELA subtype. The UBE2C and UBE2S enzymes are associated with the ubiquitin ligase anaphase promoting complex (APC/c), which regulates the degradation of substrates associated with cell cycle progression, whereas UBE2I is a Sumo-conjugating enzyme. Additionally, elevated in ST_EPN_RELA were genes for the E3 ligase and histone deacetylase HDAC4 and the F-box cullin ring ligase adaptor FBX031. Cluster analysis demonstrated several genes encoding E3 ligases and their substrate adaptors as EPN subtype specific genetic markers. The most significant Reactome Pathways associated with differentially expressed genes for E3 ligases and their adaptors included antigen presentation, neddylation, sumoylation, and the APC/c complex. Our analysis provides several UPS associated factors that may be attractive markers and future therapeutic targets for the subtype-specific treatment of EPN patients.

Slow Off-Rate Modified Aptamer (SOMAmer) Proteomic Analysis of Patient-Derived Malignant Glioma Identifies Distinct Cellular Proteomes.

Malignant gliomas derive from brain glial cells and represent >75% of primary brain tumors. This includes anaplastic astrocytoma (grade III; AS), the most common and fatal glioblastoma multiforme (grade IV; GBM), and oligodendroglioma (ODG). We have generated patient-derived AS, GBM, and ODG cell models to study disease mechanisms and test patient-centered therapeutic strategies. We have used an aptamer-based high-throughput SOMAscan® 1.3K assay to determine the proteomic profiles of 1307 different analytes. SOMAscan® proteomes of AS and GBM self-organized into closely adjacent proteomes which were clearly distinct from ODG proteomes. GBM self-organized into four proteomic clusters of which SOMAscan® cluster 4 proteome predicted a highly inter-connected proteomic network. Several up- and down-regulated proteins relevant to glioma were successfully validated in GBM cell isolates across different SOMAscan® clusters and in corresponding GBM tissues. Slow off-rate modified aptamer proteomics is an attractive analytical tool for rapid proteomic stratification of different malignant gliomas and identified cluster-specific SOMAscan® signatures and functionalities in patient GBM cells.

Platinum (IV) coiled coil nanotubes selectively kill human glioblastoma cells.

Malignant glioma are often fatal and pose a significant therapeutic challenge. Here we have employed α-helical right handed coiled coils (RHCC) which self-assemble into tetrameric nanotubes that stably associate with platinum (Pt) (IV) compound. This Pt(IV)-RHCC complex showed superior in vitro and in vivo toxicity in human malignant glioma cells at up to 5 fold lower platinum concentrations when compared to free Pt(IV). Pt(IV)-RHCC nanotubes activated multiple cell death pathways in GB cells without affecting astrocytes in vitro or causing damage to normal mouse brain. This Pt(IV)-RHCC nanotubes may serve as a promising new therapeutic tool for low dose Pt(IV) prodrug application for highly efficient and selective treatment of human brain tumors. FROM THE CLINICAL EDITOR: The prognosis of malignant glioma remains poor despite medical advances. Platinum, one of the chemotherapeutic agents used, has significant systemic side effects. In this article, the authors employed α-helical right handed coiled coil (RHCC) protein nanotubes as a carrier for cisplatin. It was shown that the new compound achieved higher tumor kill rate but lower toxicity to normal cells and thus may hold promise to be a highly efficient treatment for the future.

C1q-tumour necrosis factor-related protein 8 (CTRP8) is a novel interaction partner of relaxin receptor RXFP1 in human brain cancer cells.

We report a novel ligand-receptor system composed of the leucine-rich G-protein-coupled relaxin receptor, RXFP1, and the C1q-tumour necrosis factor-related protein 8 (CTRP8) in human primary brain cancer, a tumour entity devoid of the classical RXFP1 ligands, RLN1-3. In structural homology studies and computational docking experiments we delineated the N-terminal region of the globular C1q region of CTRP8 and the leucine-rich repeat units 7 and 8 of RXFP1 to mediate this new ligand-receptor interaction. CTRP8 secreted from HEK293T cells, recombinant human (rh) CTRP8, and short synthetic peptides derived from the C1q globular domain of human CTRP8 caused the activation of RXFP1 as determined by elevated intracellular cAMP levels and the induction of a marked pro-migratory phenotype in established glioblastoma (GB) cell lines and primary cells from GB patients. Employing a small competitor peptide, we were able to disrupt the CTRP8-RXFP1-induced increased GB motility. The CTRP8-RXFP1-mediated migration in GB cells involves the activation of PI3K and specific protein kinase C pathways and the increased production/secretion of the potent lysosomal protease cathepsin B (cathB), a known prognostic marker of GB. Specific inhibition of CTRP8-induced cathB activity effectively blocked the ability of primary GB to invade laminin matrices. Finally, co-immunoprecipitation studies revealed the direct interaction of human CTRP8 with RXFP1. Our results support a therapeutic approach in GB aimed at targeting multiple steps of the CTRP8-RXFP1 signalling pathway by a combined inhibitor and peptide-based strategy to block GB dissemination within the brain.

Overcoming brain-derived therapeutic resistance in HER2+ breast cancer brain metastasis.

Brain metastasis of HER2+ breast cancer occurs in about 50% of all women with metastatic HER2+ breast cancer and confers poor prognosis for patients. Despite effective HER2-targeted treatments of peripheral HER2+ breast cancer with Trastuzumab +/-HER2 inhibitors, limited brain permeability renders these treatments inefficient for HER2+ breast cancer brain metastasis (BCBM). The scarcity of suitable patient-derived in-vivo models for HER2+ BCBM has compromised the study of molecular mechanisms that promote growth and therapeutic resistance in brain metastasis. We have generated and characterized new HER2+ BCBM cells (BCBM94) isolated from a patient HER2+ brain metastasis. Repeated hematogenic xenografting of BCBM94 consistently generated BCBM in mice. The clinically used receptor tyrosine kinase inhibitor (RTKi) Lapatinib blocked phosphorylation of all ErbB1-4 receptors and induced the intrinsic apoptosis pathway in BCBM94. Neuregulin-1 (NRG1), a ligand for ErbB3 and ErbB4 that is abundantly expressed in the brain, was able to rescue Lapatinib-induced apoptosis and clonogenic ability in BCBM94 and in HER2+ BT474. ErbB3 was essential to mediate the NRG1-induced survival pathway that involved PI3K-AKT signalling and the phosphorylation of BAD at serine 136 to prevent apoptosis. High throughput RTKi screening identified the brain penetrable Poziotinib as highly potent compound to reduce cell viability in HER2+ BCBM in the presence of NRG1. Successful in-vivo ablation of BCBM94- and BT474-derived HER2+ brain tumors was achieved upon two weeks of treatment with Poziotinib. MRI revealed BCBM remission upon poziotinib, but not with Lapatinib treatment. In conclusion, we have established a new patient-derived HER2+ BCBM in-vivo model and identified Poziotinib as highly efficacious RTKi with excellent brain penetrability that abrogated HER2+ BCBM brain tumors in our mouse models.

Characterization of HMGA2 variants expands the spectrum of Silver-Russell syndrome.

Silver-Russell syndrome (SRS) is a heterogeneous disorder characterized by intrauterine and postnatal growth retardation. HMGA2 variants are a rare cause of SRS and its functional role in human linear growth is unclear. Patients with suspected SRS negative for 11p15LOM/mUPD7 underwent whole-exome and/or targeted-genome sequencing. Mutant HMGA2 protein expression and nuclear localization were assessed. Two Hmga2-knockin mouse models were generated. Five clinical SRS patients harbored HMGA2 variants with differing functional impacts: 2 stop-gain nonsense variants (c.49G>T, c.52C>T), c.166A>G missense variant, and 2 frameshift variants (c.144delC, c.145delA) leading to an identical, extended-length protein. Phenotypic features were highly variable. Nuclear localization was reduced/absent for all variants except c.166A>G. Homozygous knockin mice recapitulating the c.166A>G variant (Hmga2K56E) exhibited a growth-restricted phenotype. An Hmga2Ter76-knockin mouse model lacked detectable full-length Hmga2 protein, similarly to patient 3 and 5 variants. These mice were infertile, with a pygmy phenotype. We report a heterogeneous group of individuals with SRS harboring variants in HMGA2 and describe the first Hmga2 missense knockin mouse model (Hmga2K56E) to our knowledge causing a growth-restricted phenotype. In patients with clinical features of SRS but negative genetic screening, HMGA2 should be included in next-generation sequencing testing approaches.

Human C1q Tumor Necrosis Factor 8 (CTRP8) defines a novel tryptase+ mast cell subpopulation in the prostate cancer microenvironment.

The adipokine C1q Tumor Necrosis Factor 8 (CTRP8) is the least known member of the 15 CTRP proteins and a ligand of the relaxin receptor RXFP1. We previously demonstrated the ability of the CTRP8-RXFP1 interaction to promote motility, matrix invasion, and drug resistance. The lack of specific tools to detect CTRP8 protein severely limits our knowledge on CTRP8 biological functions in normal and tumor tissues. Here, we have generated and characterized the first specific antiserum to human CTRP8 which identified CTRP8 as a novel marker of tryptase+ mast cells (MCT) in normal human tissues and in the prostate cancer (PC) microenvironment. Using human PC tissue microarrays composed of neoplastic and corresponding tumor-adjacent prostate tissues, we have identified a significantly higher number of CTRP8+ MCT in the peritumor versus intratumor compartment of PC tissues of Gleason scores 6 and 7. Higher numbers of CTRP8+ MCT correlated with the clinical parameter of biochemical recurrence. We showed that the human MC line ROSAKIT WT expressed RXFP1 transcripts and responded to CTRP8 treatment with a small but significant increase in cell proliferation. Like the cognate RXFP1 ligand RLN-2 and the small molecule RXFP1 agonist ML-290, CTRP8 reduced degranulation of ROSAKIT WT MC stimulated by the Ca2+-ionophore A14187. In conclusion, this is the first report to identify the RXFP1 agonist CTRP8 as a novel marker of MCT and autocrine/paracrine oncogenic factor within the PC microenvironment.

Novel CTRP8-RXFP1-JAK3-STAT3 axis promotes Cdc42-dependent actin remodeling for enhanced filopodia formation and motility in human glioblastoma cells.

C1q tumor necrosis factor-related peptide 8 (CTRP8) is the least studied member of the C1Q-TNF-related peptide family. We identified CTRP8 as a ligand of the G protein-coupled receptor relaxin family peptide receptor 1 (RXFP1) in glioblastoma multiforme (GBM). The CTRP8-RXFP1 ligand-receptor system protects human GBM cells against the DNA-alkylating damage-inducing temozolomide (TMZ), the drug of choice for the treatment of patients with GBM. The DNA protective role of CTRP8 was dependent on a functional RXFP1-STAT3 signaling cascade and targeted the monofunctional glycosylase N-methylpurine DNA glycosylase (MPG) for more efficient base excision repair of TMZ-induced DNA-damaged sites. CTRP8 also improved the survival of GBM cells by upregulating anti-apoptotic BCl-2 and BCL-XL. Here, we have identified Janus-activated kinase 3 (JAK3) as a novel member of a novel CTRP8-RXFP1-JAK3-STAT3 signaling cascade that caused an increase in cellular protein content and activity of the small Rho GTPase Cdc42. This is associated with significant F-actin remodeling and increased GBM motility. Cdc42 was critically important for the upregulation of the actin nucleation complex N-Wiskott-Aldrich syndrome protein/Arp3/4 and actin elongation factor profilin-1. The activation of the RXFP1-JAK3-STAT3-Cdc42 axis by both RXFP1 agonists, CTRP8 and relaxin-2, caused extensive filopodia formation. This coincided with enhanced activity of ezrin, a key factor in tethering F-actin to the plasma membrane, and inhibition of the actin filament severing activity of cofilin. The F-actin remodeling and pro-migratory activities promoted by the novel RXFP1-JAK3-STAT3-Cdc42 axis were blocked by JAK3 inhibitor tofacitinib and STAT3 inhibitor STAT3 inhibitor VI. This provides a new rationale for the design of JAK3 and STAT3 inhibitors with better brain permeability for clinical treatment of the pervasive brain invasiveness of GBM.

LINE-1 methylation status of endogenous DNA double-strand breaks.

DNA methylation and the repair of DNA double-strand breaks (DSBs) are important processes for maintaining genomic integrity. Although DSBs can be produced by numerous agents, they also occur spontaneously as endogenous DSBs (EDSBs). In this study, we evaluated the methylation status of EDSBs to determine if there is a connection between DNA methylation and EDSBs. We utilized interspersed repetitive sequence polymerase chain reaction (PCR), ligation-mediated PCR and combined bisulfite restriction analysis to examine the extent of EDSBs and methylation at long interspersed nuclear element-1 (LINE-1) sequences nearby EDSBs. We tested normal white blood cells and several cell lines derived from epithelial cancers and leukemias. Significant levels of EDSBs were detectable in all cell types. EDSBs were also found in both replicating and non-replicating cells. We found that EDSBs contain higher levels of methylation than the cellular genome. This hypermethylation is replication independent and the methylation was present in the genome at the location prior to the DNA DSB. The differences in methylation levels between EDSBs and the rest of the genome suggests that EDSBs are differentially processed, by production, end-modification, or repair, depending on the DNA methylation status.

Emerging roles for the relaxin/RXFP1 system in cancer therapy.

A role for the hormone relaxin in cancer was described well before the receptor was identified. Relaxin predominantly increases the growth and invasive potential in cancers of different origins. However, relaxin was also shown to promote cell differentiation and to act in a dose-and time-dependent manner in different cancer cell models used. Following the discovery of the relaxin like family peptide receptor 1 (RXFP1) as the cellular receptor for RLN1 and RLN2, research has focussed on the ligand interaction with the large extracellular domain of RXFP1 and resulting molecular signaling mechanisms. RXFP1 activation mediates anti-apoptotic functions, angiogenesis and chemoresistance in cancer cells. This minireview summarizes the known biological functions of RXFP1 activation in different cancer entities in-vitro and in-vivo and outlines possible mechanisms to therapeutically address the relaxin-RXFP1 system in cancer cells.

HMGA2 as a functional antagonist of PARP1 inhibitors in tumor cells.

Poly(ADP-ribose) polymerase 1 inhibitors alone or in combination with DNA damaging agents are promising clinical drugs in the treatment of cancer. However, there is a need to understand the molecular mechanisms of resistance to PARP1 inhibitors. Expression of HMGA2 in cancer is associated with poor prognosis for patients. Here, we investigated the novel relationship between HMGA2 and PARP1 in DNA damage-induced PARP1 activity. We used human triple-negative breast cancer and fibrosarcoma cell lines to demonstrate that HMGA2 colocalizes and interacts with PARP1. High cellular HMGA2 levels correlated with increased DNA damage-induced PARP1 activity, which was dependent on functional DNA-binding AT-hook domains of HMGA2. HMGA2 inhibited PARP1 trapping to DNA and counteracted the cytotoxic effect of PARP inhibitors. Consequently, HMGA2 decreased caspase 3/7 induction and increased cell survival upon treatment with the alkylating methyl methanesulfonate alone or in combination with the PARP inhibitor AZD2281 (olaparib). HMGA2 increased mitochondrial oxygen consumption rate and spare respiratory capacity and increased NAMPT levels, suggesting metabolic support for enhanced PARP1 activity upon DNA damage. Our data showed that expression of HMGA2 in cancer cells reduces sensitivity to PARP inhibitors and suggests that targeting HMGA2 in combination with PARP inhibition may be a promising new therapeutic approach.

C1q/TNF-related peptide 8 (CTRP8) promotes temozolomide resistance in human glioblastoma.

The C1q/TNF-related peptide 8 (CTRP8) has recently emerged as a novel ligand of the G protein-coupled receptor RXFP1 in the fatal brain tumor glioblastoma (GBM). We previously demonstrated that the CTRP8-RXFP1 ligand-receptor system promotes motility and matrix invasion of patient GBM and U87 MG cells by specific phosphorylation of PI3 kinase and protein kinase C. Here, we demonstrate a novel role for CTRP8 in protecting human GBM cells against the DNA alkylating damage of temozolomide (TMZ), the standard chemotherapy drug used to treat GBM. This DNA protective role of CTRP8 required a functional RXFP1-STAT3 signaling cascade in GBM cells. We identified N-methylpurine DNA glycosylase (MPG), a monofunctional glycosylase that initiates base excision repair pathway by generating an apurinic/apyrimidinic (AP) site, as a new CTRP8-RXFP1-STAT3 target in GBM. Upon TMZ exposure, treatment with CTRP8 reduced the formation of AP sites and double-strand DNA breaks in GBM cells. This CTRP8 effect was independent of cellular MGMT levels and was associated with decreased caspase 3/7 activity and increased survival of human GBM. CTRP8-induced RXFP1 activation caused an increase in cellular protein levels of the anti-apoptotic Bcl members and STAT3 targets Bcl-2 and Bcl-XL in human GBM. Collectively, our results demonstrate a novel multipronged and clinically relevant mechanism by which the CTRP8-RXFP1 ligand-receptor system exerts a DNA protective function against TMZ chemotherapeutic stress in GBM. This CTRP8-RXFP1-STAT3 axis is a novel determinant of TMZ responsiveness/chemoresistance and an emerging new drug target for improved treatment of human GBM.

Dovitinib enhances temozolomide efficacy in glioblastoma cells.

The multikinase inhibitor and FDA-approved drug dovitinib (Dov) crosses the blood-brain barrier and was recently used as single drug application in clinical trials for GB patients with recurrent disease. The Dov-mediated molecular mechanisms in GB cells are unknown. We used GB patient cells and cell lines to show that Dov downregulated the stem cell protein Lin28 and its target high-mobility group protein A2 (HMGA2). The Dov-induced reduction in pSTAT3Tyr705 phosphorylation demonstrated that Dov negatively affects the STAT3/LIN28/Let-7/HMGA2 regulatory axis in GB cells. Consistent with the known function of LIN28 and HMGA2 in GB self-renewal, Dov reduced GB tumor sphere formation. Dov treatment also caused the downregulation of key base excision repair factors and O6 -methylguanine-DNA-methyltransferase (MGMT), which are known to have important roles in the repair of temozolomide (TMZ)-induced alkylating DNA damage. Combined Dov/TMZ treatment enhanced TMZ-induced DNA damage as quantified by nuclear γH2AX foci and comet assays, and increased GB cell apoptosis. Pretreatment of GB cells with Dov ('Dov priming') prior to TMZ treatment reduced GB cell viability independent of p53 status. Sequential treatment involving 'Dov priming' and alternating treatment cycles with TMZ and Dov substantially reduced long-term GB cell survival in MGMT+ patient GB cells. Our results may have immediate clinical implications to improve TMZ response in patients with LIN28+ /HMGA2+ GB, independent of their MGMT methylation status.

Structural commonality of C1q TNF-related proteins and their potential to activate relaxin/insulin-like family peptide receptor 1 signalling pathways in cancer cells.

We established the role of the GPCR relaxin/insulin-like family peptide receptor 1 (RXFP1 receptor) as a novel active receptor in human glioblastoma (GB), a fatal brain tumour. We identified C1q/TNF-related protein 8 (CTRP8) as a novel agonist of the RXFP1 receptor. CTRP8 enhanced the motility and matrix invasion of GB, and this involved PKC-mediated up-regulation of cathepsin B, a marker for poor prognosis in GB patients. We conclude that the absence of relaxin isoforms does not preclude the activation of the RXFP1 receptor, as the least known member of the CTRP family, CTRP8, can effectively target and activate RXFP1 receptors. LINKED ARTICLES: This article is part of a themed section on Recent Progress in the Understanding of Relaxin Family Peptides and their Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.10/issuetoc.

RXFP1 is Targeted by Complement C1q Tumor Necrosis Factor-Related Factor 8 in Brain Cancer.

The relaxin-like RXFP1 ligand-receptor system has important functions in tumor growth and tissue invasion. Recently, we have identified the secreted protein, CTRP8, a member of the C1q/tumor necrosis factor-related protein (CTRP) family, as a novel ligand of the relaxin receptor, RXFP1, with functions in brain cancer. Here, we review the role of CTRP members in cancers cells with particular emphasis on CTRP8 in glioblastoma.

PDK2-mediated alternative splicing switches Bnip3 from cell death to cell survival.

Herein we describe a novel survival pathway that operationally links alternative pre-mRNA splicing of the hypoxia-inducible death protein Bcl-2 19-kD interacting protein 3 (Bnip3) to the unique glycolytic phenotype in cancer cells. While a full-length Bnip3 protein (Bnip3FL) encoded by exons 1-6 was expressed as an isoform in normal cells and promoted cell death, a truncated spliced variant of Bnip3 mRNA deleted for exon 3 (Bnip3Δex3) was preferentially expressed in several human adenocarcinomas and promoted survival. Reciprocal inhibition of the Bnip3Δex3/Bnip3FL isoform ratio by inhibiting pyruvate dehydrogenase kinase isoform 2 (PDK2) in Panc-1 cells rapidly induced mitochondrial perturbations and cell death. The findings of the present study reveal a novel survival pathway that functionally couples the unique glycolytic phenotype in cancer cells to hypoxia resistance via a PDK2-dependent mechanism that switches Bnip3 from cell death to survival. Discovery of the survival Bnip3Δex3 isoform may fundamentally explain how certain cells resist Bnip3 and avert death during hypoxia.

Mechanisms of therapeutic resistance in cancer (stem) cells with emphasis on thyroid cancer cells.

The two main reasons for death of cancer patients, tumor recurrence and metastasis, are multi-stage cellular processes that involve increased cell plasticity and coincide with elevated resistance to anti-cancer treatments. Epithelial-to-mesenchymal transition (EMT) is a key contributor to metastasis in many cancer types, including thyroid cancer and is known to confer stem cell-like properties onto cancer cells. This review provides an overview of molecular mechanisms and factors known to contribute to cancer cell plasticity and capable of enhancing cancer cell resistance to radio- and chemotherapy. We elucidate the role of DNA repair mechanisms in contributing to therapeutic resistance, with a special emphasis on thyroid cancer. Next, we explore the emerging roles of autophagy and damage-associated molecular pattern responses in EMT and chemoresistance in tumor cells. Finally, we demonstrate how cancer cells, including thyroid cancer cells, can highjack the oncofetal nucleoprotein high-mobility group A2 to gain increased transformative cell plasticity, prevent apoptosis, and enhance metastasis of chemoresistant tumor cells.

INSL5 is a novel marker for human enteroendocrine cells of the large intestine and neuroendocrine tumours.

We report for the first time the distribution of human INSL5 and its cognate leucine rich G-protein coupled receptor RXFP4 in the large intestine and in neuroendocrine/carcinoid tissues. Immunoreactive INSL5 was uniquely expressed by enteroendocrine cells (EECs) located within the colonic mucosa, whereas colonocytes were immunopositive for RXFP4. INSL5+ and RXFP4+ cells were also detected in human neuroendocrine/carcinoid tissues. We employed a recently described Insl5 knockout mouse model and 2 mouse models of induced colitis to address the relevance of Insl5 in EEC development and in acute inflammation of the colon. We identified INSL5 as a specific marker for synaptophysin+ EECs in the mucosa of the normal human and mouse colon. Insl5 was not essential for the development of mouse synaptophysin+ EECs. The mouse models of chemically induced colitis (dextran sulfate sodium and dinitrobenzene-sulfonic acid) failed to show changes in the numbers of Insl5+ EECs at inflammatory sites during the acute phase of colitis. In conclusion, we showed that INSL5 is a novel marker of colorectal EECs and provide first evidence for the presence of a potentially autocrine/paracrine INSL5-RXFP4 signaling system in the normal human and mouse colon and in rare human neuroendocrine tumours.

INSL5-deficient mice display an alteration in glucose homeostasis and an impaired fertility.

Insulin-like factor 5 (INSL5), a member of the insulin superfamily, is expressed in the colorectum and hypothalamus. To facilitate studies into the role of INSL5, we generated Insl5(-/-) mice by gene targeting. Insl5(-/-) mice were born in the expected Mendelian ratio, reached normal body weight, but displayed impaired male and female fertility that are due to marked reduction in sperm motility and irregular length of the estrous cycle. Furthermore, Insl5(-/-) mice showed impairment in glucose homeostasis with characteristic elevation of serum glucose levels at an advanced age. Glucose and insulin tolerance tests revealed that the increased blood glucose in Insl5(-/-) mice was due to glucose intolerance resulting from reduced insulin secretion. Morphometric and immunohistological analyses revealed that the Insl5(-/-) mice had markedly reduced average islets area and ß-cell numbers. Furthermore, immunohistochemistry showed the expression of INSL5 in enteroendocrine cells in the colorectal epithelium and the presence of its putative receptor relaxin family peptide receptor 4 in pancreatic islet cells. These results suggest the potential role of INSL5 signaling in the regulation of insulin secretion and ß-cell homeostasis.