Serine protease PRSS55 is crucial for male mouse fertility via affecting sperm migration and sperm–egg binding

Testis-specific PRSS55 is a highly conserved chymotrypsin-like serine protease among mammalian species. So far, the physiological function of PRSS55 remains unknown. Here, we show that PRSS55 is a GPI-anchored membrane protein, specifically expressed in adult mouse testis and mainly observed in the luminal side of seminiferous tubules and sperm acrosome. Mice deficient for Prss55 develop male infertile with normal reproduction-related parameters observed. Interestingly, in vivo fertilization rate of Prss55^?/? males is dramatically decreased, possibly due to incapable migration of Prss55^?/? sperm from uterus into oviduct. However, in vitro fertilization rate has no difference between two genotypes although Prss55^?/? sperm presents defective recognition/binding to zona-intact or zona-free oocytes. Further study reveals that mature ADAM3 is almost undetectable in Prss55^?/? sperm, while precursor ADAM3 remains unchanged in the testis. However, it is shown that ADAM3 has no interaction with PRSS55 by immunoprecipitation with anti-PRSS55 antibody. The expression levels of several proteins known to be related to the observed phenotypes remain comparable between wt and Prss55^?/? mice. Moreover, we found that Prss55 deficiency has no effect on PRSS37 or vice versa albeit two mutant males share almost the same phenotypes. Microarray analysis reveals a total of 72 differentially expressed genes in Prss55^?/? testis, most of which are associated with cellular membrane and organelle organization, protein transport and complex assembly, and response to stimulus and signaling. In conclusion, we have demonstrated that PRSS55 plays vital roles in regulating male fertility of mice, including in vivo sperm migration and in vitro sperm–egg interaction, possibly by affecting the maturation of ADAM3 in sperm and the expression of multiple genes in testis.     

Ablation of kallikrein 7 (KLK7) in adipose tissue ameliorates metabolic consequences of high fat diet-induced obesity by counteracting adipose tissue inflammation in vivo

Vaspin is an adipokine which improves glucose metabolism and insulin sensitivity in obesity. Kallikrein 7 (KLK7) is the first known protease target inhibited by vaspin and a potential target for the treatment of metabolic disorders. Here, we tested the hypothesis that inhibition of KLK7 in adipose tissue may beneficially affect glucose metabolism and adipose tissue function. Therefore, we have inactivated the Klk7 gene in adipose tissue using conditional gene-targeting strategies in mice. Klk7-deficient mice (ATKlk7 ^?/?) exhibited less weight gain, predominant expansion of subcutaneous adipose tissue and improved whole body insulin sensitivity under a high fat diet (HFD). ATKlk7 ^?/? mice displayed higher energy expenditure and food intake, most likely due to altered adipokine secretion including lower circulating leptin. Pro-inflammatory cytokine expression was significantly reduced in combination with an increased percentage of alternatively activated (anti-inflammatory) M2 macrophages in epigonadal adipose tissue of ATKlk7 ^?/?. Taken together, by attenuating adipose tissue inflammation, altering adipokine secretion and epigonadal adipose tissue expansion, Klk7 deficiency in adipose tissue partially ameliorates the adverse effects of HFD-induced obesity. In summary, we provide first evidence for a previously unrecognized role of KLK7 in adipose tissue with effects on whole body energy expenditure and insulin sensitivity.     

Dicer generates a regulatory microRNA network in smooth muscle cells that limits neointima formation during vascular repair

MicroRNAs (miRNAs) coordinate vascular repair by regulating injury-induced gene expression in vascular smooth muscle cells (SMCs) and promote the transition of SMCs from a contractile to a proliferating phenotype. However, the effect of miRNA expression in SMCs on neointima formation is unclear. Therefore, we studied the role of miRNA biogenesis by Dicer in SMCs in vascular repair. Following wire-induced injury to carotid arteries of Apolipoprotein E knockout (Apoe ^?/?) mice, miRNA microarray analysis revealed that the most significantly regulated miRNAs, such as miR-222 and miR-21-3p, were upregulated. Conditional deletion of Dicer in SMCs increased neointima formation by reducing SMC proliferation in Apoe ^?/? mice, and decreased mainly the expression of miRNAs, such as miR-147 and miR-100, which were not upregulated following vascular injury. SMC-specific deletion of Dicer promoted growth factor and inflammatory signaling and regulated a miRNA–target interaction network in injured arteries that was enriched in anti-proliferative miRNAs. The most connected miRNA in this network was miR-27a-3p [e.g., with Rho guanine nucleotide exchange factor 26 (ARHGEF26)], which was expressed in medial and neointimal SMCs in a Dicer-dependent manner. In vitro, miR-27a-3p suppresses ARHGEF26 expression and inhibits SMC proliferation by interacting with a conserved binding site in the 3′ untranslated region of ARHGEF26 mRNA. We propose that Dicer expression in SMCs plays an essential role in vascular repair by generating anti-proliferative miRNAs, such as miR-27a-3p, to prevent vessel stenosis due to exaggerated neointima formation.     

The adhesion receptor GPR56 is activated by extracellular matrix collagen III to improve β-cell function

Aims

G-protein coupled receptor 56 (GPR56) is the most abundant islet-expressed G-protein coupled receptor, suggesting a potential role in islet function. This study evaluated islet expression of GPR56 and its endogenous ligand collagen III, and their effects on β-cell function.

Methods

GPR56 and collagen III expression in mouse and human pancreas sections was determined by fluorescence immunohistochemistry. Effects of collagen III on β-cell proliferation, apoptosis, intracellular calcium ([Ca²⁺]_i) and insulin secretion were determined by cellular BrdU incorporation, caspase 3/7 activities, microfluorimetry and radioimmunoassay, respectively. The role of GPR56 in islet vascularisation and innervation was evaluated by immunohistochemical staining for CD31 and TUJ1, respectively, in pancreases from wildtype (WT) and Gpr56^?/? mice, and the requirement of GPR56 for normal glucose homeostasis was determined by glucose tolerance tests in WT and Gpr56^?/? mice.

Results

Immunostaining of mouse and human pancreases revealed that GPR56 was expressed by islet β-cells while collagen III was confined to the peri-islet basement membrane and islet capillaries. Collagen III protected β-cells from cytokine-induced apoptosis, triggered increases in [Ca²⁺]_i and potentiated glucose-induced insulin secretion from WT islets but not from Gpr56^?/? islets. Deletion of GPR56 did not affect glucose-induced insulin secretion in vitro and it did not impair glucose tolerance in adult mice. GPR56 was not required for normal islet vascularisation or innervation.

Conclusion

We have demonstrated that collagen III improves islet function by increasing insulin secretion and protecting against apoptosis. Our data suggest that collagen III may be effective in optimising islet function to improve islet transplantation outcomes, and GPR56 may be a target for the treatment of type 2 diabetes.

     

A threshold of transmembrane potential is required for mitochondrial dynamic balance mediated by DRP1 and OMA1

As an organellar network, mitochondria dynamically regulate their organization via opposing fusion and fission pathways to maintain bioenergetic homeostasis and contribute to key cellular pathways. This dynamic balance is directly linked to bioenergetic function: loss of transmembrane potential across the inner membrane (Δψ _m) disrupts mitochondrial fission/fusion balance, causing fragmentation of the network. However, the level of Δψ _m required for mitochondrial dynamic balance, as well as the relative contributions of fission and fusion pathways, have remained unclear. To explore this, mitochondrial morphology and Δψ _m were examined via confocal imaging and tetramethyl rhodamine ester (TMRE) flow cytometry, respectively, in cultured 143B osteosarcoma cells. When normalized to the TMRE value of untreated 143B cells as 100%, both genetic (mtDNA-depleted ρ⁰) and pharmacological [carbonyl cyanide m-chlorophenyl hydrazone (CCCP)-treated] cell models below 34% TMRE fluorescence were unable to maintain mitochondrial interconnection, correlating with loss of fusion-active long OPA1 isoforms (L-OPA1). Mechanistically, this threshold is maintained by mechanistic coordination of DRP1-mediated fission and OPA1-mediated fusion: cells lacking either DRP1 or the OMA1 metalloprotease were insensitive to loss of Δψ _m, instead maintaining an obligately fused morphology. Collectively, these findings demonstrate a mitochondrial ‘tipping point’ threshold mediated by the interaction of Δψ _m with both DRP1 and OMA1; moreover, DRP1 appears to be required for effective OPA1 maintenance and processing, consistent with growing evidence for direct interaction of fission and fusion pathways. These results suggest that Δψ _m below threshold coordinately activates both DRP1-mediated fission and OMA1 cleavage of OPA1, collapsing mitochondrial dynamic balance, with major implications for a range of signaling pathways and cellular life/death events.     

Retinoic acid regulates avian lung branching through a molecular network

Retinoic acid (RA) is of major importance during vertebrate embryonic development and its levels need to be strictly regulated otherwise congenital malformations will develop. Through the action of specific nuclear receptors, named RAR/RXR, RA regulates the expression of genes that eventually influence proliferation and tissue patterning. RA has been described as crucial for different stages of mammalian lung morphogenesis, and as part of a complex molecular network that contributes to precise organogenesis; nonetheless, nothing is known about its role in avian lung development. The current report characterizes, for the first time, the expression pattern of RA signaling members (stra6, raldh2, raldh3, cyp26a1, rarα, and rarβ) and potential RA downstream targets (sox2, sox9, meis1, meis2, tgfβ2, and id2) by in situ hybridization. In the attempt of unveiling the role of RA in chick lung branching, in vitro lung explants were performed. Supplementation studies revealed that RA stimulates lung branching in a dose-dependent manner. Moreover, the expression levels of cyp26a1, sox2, sox9, rarβ, meis2, hoxb5, tgfβ2, id2, fgf10, fgfr2, and shh were evaluated after RA treatment to disclose a putative molecular network underlying RA effect. In situ hybridization analysis showed that RA is able to alter cyp26a1, sox9, tgfβ2, and id2 spatial distribution; to increase rarβ, meis2, and hoxb5 expression levels; and has a very modest effect on sox2, fgf10, fgfr2, and shh expression levels. Overall, these findings support a role for RA in the proximal–distal patterning and branching morphogenesis of the avian lung and reveal intricate molecular interactions that ultimately orchestrate branching morphogenesis.     

<Emphasis Type="Italic">C</Emphasis>-<Emphasis Type="Italic">Raf</Emphasis> deficiency leads to hearing loss and increased noise susceptibility

The family of RAF kinases transduces extracellular information to the nucleus, and their activation is crucial for cellular regulation on many levels, ranging from embryonic development to carcinogenesis. B-RAF and C-RAF modulate neurogenesis and neuritogenesis during chicken inner ear development. C-RAF deficiency in humans is associated with deafness in the rare genetic insulin-like growth factor 1 (IGF-1), Noonan and Leopard syndromes. In this study, we show that RAF kinases are expressed in the developing inner ear and in adult mouse cochlea. A homozygous C-Raf deletion in mice caused profound deafness with no evident cellular aberrations except for a remarkable reduction of the K⁺ channel Kir4.1 expression, a trait that suffices as a cause of deafness. To explore the role of C-Raf in cellular protection and repair, heterozygous C-Raf ^+/? mice were exposed to noise. A reduced C-RAF level negatively affected hearing preservation in response to noise through mechanisms involving the activation of JNK and an exacerbated apoptotic response. Taken together, these results strongly support a role for C-RAF in hearing protection.     

<Emphasis Type="Italic">Yersinia pseudotuberculosis</Emphasis> supports Th17 differentiation and limits de novo regulatory T cell induction by directly interfering with T cell receptor signaling

Adaptive immunity critically contributes to control acute infection with enteropathogenic Yersinia pseudotuberculosis; however, the role of CD4⁺ T cell subsets in establishing infection and allowing pathogen persistence remains elusive. Here, we assessed the modulatory capacity of Y. pseudotuberculosis on CD4⁺ T cell differentiation. Using in vivo assays, we report that infection with Y. pseudotuberculosis resulted in enhanced priming of IL-17-producing T cells (Th17 cells), whereas induction of Foxp3⁺ regulatory T cells (Tregs) was severely disrupted in gut-draining mesenteric lymph nodes (mLNs), in line with altered frequencies of tolerogenic and proinflammatory dendritic cell (DC) subsets within mLNs. Additionally, by using a DC-free in vitro system, we could demonstrate that Y. pseudotuberculosis can directly modulate T cell receptor (TCR) downstream signaling within naïve CD4⁺ T cells and Tregs via injection of effector molecules through the type III secretion system, thereby affecting their functional properties. Importantly, modulation of naïve CD4⁺ T cells by Y. pseudotuberculosis resulted in an enhanced Th17 differentiation and decreased induction of Foxp3⁺ Tregs in vitro. These findings shed light to the adjustment of the Th17-Treg axis in response to acute Y. pseudotuberculosis infection and highlight the direct modulation of CD4⁺ T cell subsets by altering their TCR downstream signaling.     

Experimental verification of a conserved intronic microRNA located in the human <Emphasis Type="Italic">TrkC</Emphasis> gene with a cell type-dependent apoptotic function

Tropomyosin receptor kinase C (TrkC) is involved in cell survival, apoptosis induction and tumorigenesis. We hypothesized that, similar to p75^NTR receptor, some of the diverse functions of TrkC could be mediated by a microRNA (miRNA) embedded within the gene. Here, we experimentally verified the expression and processing of two bioinformatically predicted miRNAs named TrkC-miR1-5p and TrkC-miR1-3p. Transfecting a DNA fragment corresponding to the TrkC-premir1 sequence in HEK293t cells caused ~300-fold elevation in the level of mature TrkC-miR1 and also a significant downregulation of its predicted target genes. Furthermore, endogenous TrkC-miR1 was detected in several cell lines and brain tumors confirming its endogenous generation. Furthermore, its orthologous miRNA was detected in developing rat brain. Accordingly, TrkC-miR1 expression was increased during the course of neural differentiation of NT2 cell, whereas its suppression attenuated NT2 differentiation. Consistent with opposite functions of TrkC, TrkC-miR1 overexpression promoted survival and apoptosis in U87 and HEK293t cell lines, respectively. In conclusion, our data report the discovery of a new miRNA with overlapping function to TrkC.     

Extracellular vesicles regulate the human osteoclastogenesis: divergent roles in discrete inflammatory arthropathies

Objective

Extracellular vesicles (EVs) are subcellular signalosomes. Although characteristic EV production is associated with numerous physiological and pathological conditions, the effect of blood-derived EVs on bone homeostasis is unknown. Herein we evaluated the role of circulating EVs on human osteoclastogenesis.

Methods

Blood samples from healthy volunteers, rheumatoid arthritis (RA) and psoriatic arthritis (PsA) patients were collected. Size-based EV sub-fractions were isolated by gravity-driven filtration and differential centrifugation. To investigate the properties of EV samples, resistive pulse sensing technique, transmission electron microscopy, flow cytometry and western blot were performed. CD14⁺ monocytes were separated from PBMCs, and stimulated with recombinant human M-CSF, RANKL and blood-derived EV sub-fractions. After 7 days, the cells were fixed and stained for tartrate-resistant acid phosphatase and counted.

Results

EVs isolated by size-based sub-fractions were characterized as either microvesicles or exosomes (EXO). Healthy (n = 11) and RA-derived (n = 12) EXOs profoundly inhibited osteoclast differentiation (70%, p < 0.01; 65%, p < 0.01, respectively). In contrast, PsA-derived (n = 10) EXOs had a stimulatory effect (75%, p < 0.05). In cross-treatment experiments where EXOs and CD14⁺ cells were interchanged between the three groups, only healthy (n = 5) and RA (n = 5)-derived EXOs inhibited (p < 0.01, respectively) the generation of osteoclasts in all groups, whereas PsA (n = 7)-derived EXOs were unable to mediate this effect.

Conclusions

Our data suggest that blood-derived EXOs are novel regulators of the human osteoclastogenesis and may offer discrete effector function in distinct inflammatory arthropathies.

     

<Emphasis Type="Italic">Cis</Emphasis>–<Emphasis Type="Italic">trans</Emphasis> interactions of cell surface receptors: biological roles and structural basis

Cell surface receptors bind ligands expressed on other cells (in trans) in order to communicate with neighboring cells. However, an increasing number of cell surface receptors are found to also interact with ligands expressed on the same cell (in cis). These observations raise questions regarding the biological role of such cis interactions. Specifically, it is important to know whether cis and trans binding have distinct functional effects and, if so, how a single cell discriminates between interactions in cis versus trans. Further, what are the structural features that allow certain cell surface receptors to engage ligand both on the same as well as on an apposed cell membrane? Here, we summarize known examples of receptors that display cis–trans binding and discuss the emerging diversity of biological roles played by these unconventional two-way interactions, along with their structural basis.     

Human endoglin as a potential new partner involved in platelet–endothelium interactions

Complex interactions between platelets and activated endothelium occur during the thrombo-inflammatory reaction at sites of vascular injuries and during vascular hemostasis. The endothelial receptor endoglin is involved in inflammation through integrin-mediated leukocyte adhesion and transmigration; and heterozygous mutations in the endoglin gene cause hereditary hemorrhagic telangiectasia type 1. This vascular disease is characterized by a bleeding tendency that is postulated to be a consequence of telangiectasia fragility rather than a platelet defect, since platelets display normal functions in vitro in this condition. Here, we hypothesize that endoglin may act as an adhesion molecule involved in the interaction between endothelial cells and platelets through integrin recognition. We find that the extracellular domain of human endoglin promotes specific platelet adhesion under static conditions and confers resistance of adherent platelets to detachment upon exposure to flow. Also, platelets adhere to confluent endothelial cells in an endoglin-mediated process. Remarkably, Chinese hamster ovary cells ectopically expressing the human αIIbβ3 integrin acquire the capacity to adhere to myoblast transfectants expressing human endoglin, whereas platelets from Glanzmann’s thrombasthenia patients lacking the αIIbβ3 integrin are defective for endoglin-dependent adhesion to endothelial cells. Furthermore, the bleeding time, but not the prothrombin time, is significantly prolonged in endoglin-haplodeficient (Eng ^+/?) mice compared to Eng ^+/+ animals. These results suggest a new role for endoglin in αIIbβ3 integrin-mediated adhesion of platelets to the endothelium, and may provide a better understanding on the basic cellular mechanisms involved in hemostasis and thrombo-inflammatory events.     

Neural stem cells in Parkinson’s disease: a role for neurogenesis defects in onset and progression

Parkinson’s disease (PD) is the second most common neurodegenerative disorder, leading to a variety of motor and non-motor symptoms. Interestingly, non-motor symptoms often appear a decade or more before the first signs of motor symptoms. Some of these non-motor symptoms are remarkably similar to those observed in cases of impaired neurogenesis and several PD-related genes have been shown to play a role in embryonic or adult neurogenesis. Indeed, animal models deficient in Nurr1, Pitx3, SNCA and PINK1 display deregulated embryonic neurogenesis and LRRK2 and VPS35 have been implicated in neuronal development-related processes such as Wnt/β-catenin signaling and neurite outgrowth. Moreover, adult neurogenesis is affected in both PD patients and PD animal models and is regulated by dopamine and dopaminergic (DA) receptors, by chronic neuroinflammation, such as that observed in PD, and by differential expression of wild-type or mutant forms of PD-related genes. Indeed, an increasing number of in vivo studies demonstrate a role for SNCA and LRRK2 in adult neurogenesis and in the generation and maintenance of DA neurons. Finally, the roles of PD-related genes, SNCA, LRRK2, VPS35, Parkin, PINK1 and DJ-1 have been studied in NSCs, progenitor cells and induced pluripotent stem cells, demonstrating a role for some of these genes in stem/progenitor cell proliferation and maintenance. Together, these studies strongly suggest a link between deregulated neurogenesis and the onset and progression of PD and present strong evidence that, in addition to a neurodegenerative disorder, PD can also be regarded as a developmental disorder.     

Defective germinal center B-cell response and reduced arthritic pathology in microRNA-29a-deficient mice

MicroRNA (miR) are short non-coding RNA sequences of 19–24 nucleotides that regulate gene expression by binding to mRNA target sequences. The miR-29 family of miR (miR-29a, b-1, b-2 and c) is a key player in T-cell differentiation and effector function, with deficiency causing thymic involution and a more inflammatory T-cell profile. However, the relative roles of different miR-29 family members in these processes have not been dissected. We studied the immunological role of the individual members of the miR-29 family using mice deficient for miR-29a/b-1 or miR-29b-2/c in homeostasis and during collagen-induced arthritis. We found a definitive hierarchy of immunological function, with the strong phenotype of miR-29a-deficiency in thymic involution and T-cell activation being reduced or absent in miR-29c-deficient mice. Strikingly, despite elevating the Th1 and Th17 responses, loss of miR-29a conferred near-complete protection from collagen-induced arthritis (CIA), with profound defects in B-cell proliferation and antibody production. Our results identify the hierarchical structure of the miR-29 family in T-cell biology, and identify miR-29a in B cells as a potential therapeutic target in arthritis.     

miR-124-3p is a chronic regulator of gene expression after brain injury

Traumatic brain injury (TBI) initiates molecular and cellular pathologies that underlie post-injury morbidities, including hippocampus-related memory decline and epileptogenesis. Non-coding small RNAs are master regulators of gene expression with the potential to affect multiple molecular pathways. To evaluate whether hippocampal gene expression networks are chronically regulated by microRNAs after TBI, we sampled the dentate gyrus of rats with severe TBI induced by lateral fluid-percussion injury 3 months earlier. Ingenuity pathway analysis revealed 30 upregulated miR-124-3p targets, suggesting that miR-124-3p is downregulated post-TBI (z-score?=?? 5.146, p?<?0.05). Droplet digital polymerase chain reaction (ddPCR) and in situ hybridization confirmed the chronic downregulation of miR-124-3p (p?<?0.05). Quantitative PCR analysis of two targets, Plp2 and Stat3, indicated that their upregulation correlated with the miR-124-3p downregulation (r?=?? 0.647, p?<?0.05; r?=?? 0.629, p?<?0.05, respectively). Immunohistochemical staining of STAT3 confirmed the increased protein expression. STRING analysis showed that 9 of the 30 miR-124-3p targets belonged to a STAT3 network. Reactome analysis and data mining connected the targets especially to inflammation and signal transduction. L1000CDS2 software revealed drugs (e.g., importazole, trichostatin A, and IKK-16) that could reverse the observed molecular changes. The translational value of our data was emphasized by in situ hybridization showing chronic post-traumatic downregulation of miR-124-3p in the dentate gyrus of TBI patients. Analysis of another brain injury model, status epilepticus, highlighted the fact that chronic downregulation of miR-124 is a common phenomenon after brain injury. Together, our findings indicate that miR-124-3p is a chronic modulator of molecular networks relevant to post-injury hippocampal pathologies in experimental models and in humans.     

UDP-glucose ceramide glucosyltransferase activates AKT,promoted proliferation,and doxorubicin resistance in breast cancer cells

The UDP-glucose ceramide glucosyltransferase (UGCG) is a key enzyme in the synthesis of glycosylated sphingolipids, since this enzyme generates the precursor for all complex glycosphingolipids (GSL), the GlcCer. The UGCG has been associated with several cancer-related processes such as maintaining cancer stem cell properties or multidrug resistance induction. The precise mechanisms underlying these processes are unknown. Here, we investigated the molecular mechanisms occurring after UGCG overexpression in breast cancer cells. We observed alterations of several cellular properties such as morphological changes, which enhanced proliferation and doxorubicin resistance in UGCG overexpressing MCF-7 cells. These cellular effects seem to be mediated by an altered composition of glycosphingolipid-enriched microdomains (GEMs), especially an accumulation of globotriaosylceramide (Gb3) and glucosylceramide (GlcCer), which leads to an activation of Akt and ERK1/2. The induction of the Akt and ERK1/2 signaling pathway results in an increased gene expression of multidrug resistance protein 1 (MDR1) and anti-apoptotic genes and a decrease of pro-apoptotic gene expression. Inhibition of the protein kinase C (PKC) and phosphoinositide 3 kinase (PI3K) reduced MDR1 gene expression. This study discloses how changes in UGCG expression impact several cellular signaling pathways in breast cancer cells resulting in enhanced proliferation and multidrug resistance.