Transgenic expression of Dspp partially rescued the long bone defects of Dmp1-null mice

Dentin matrix protein 1 (DMP1) and dentin sialophosphoprotein (DSPP) belong to the Small Integrin-Binding Ligand N-linked Glycoprotein (SIBLING) family. In addition to the features common to all SIBLING members, DMP1 and DSPP share several unique similarities in chemical structure, proteolytic activation and tissue localization. Mutations in, or deletion of DMP1, cause autosomal recessive hypophosphatemic rickets along with dental defects; DSPP mutations or its ablation are associated with dentinogenesis imperfecta. While the roles and functional mechanisms of DMP1 in osteogenesis have been extensively studied, those of DSPP in long bones have been studied only to a limited extent. Previous studies by our group revealed that transgenic expression of Dspp completely rescued the dentin defects of Dmp1-null (Dmp1^−/−) mice. In this investigation, we assessed the effects of transgenic Dspp on osteogenesis by analyzing the formation and mineralization of the long bones in Dmp1^−/− mice that expresses a transgene encoding full-length DSPP driven by a 3.6-kb rat Col1a1 promoter (referred as “Dmp1^−/−;Dspp-Tg mice”). We characterized the long bones of the Dmp1^−/−;Dspp-Tg mice at different ages and compared them with those from Dmp1^−/− and Dmp1^+/− (normal control) mice. Our analyses showed that the long bones of Dmp1^−/−;Dspp-Tg mice had a significant increase in cortical bone thickness, bone volume and mineral density along with a remarkable restoration of trabecular thickness compared to those of the Dmp1^−/− mice. The long bones of Dmp1^−/−;Dspp-Tg mice underwent a dramatic reduction in the amount of osteoid, significant improvement of the collagen fibrillar network, and better organization of the lacunocanalicular system, compared to the Dmp1^−/− mice. The elevated levels of biglycan, bone sialoprotein and osteopontin in Dmp1^−/− mice were also noticeably corrected by the transgenic expression of Dspp. These findings suggest that DSPP and DMP1 may function synergistically within the complex milieus of bone matrices.     

Haematopoietic TLR4 deletion attenuates perivascular brown adipose tissue inflammation in atherosclerotic mice

AimsTo investigate whether haematopoietic TLR4 deletion attenuates perivascular brown adipose tissue inflammation in atherosclerotic mice.Methods and ResultsExperiments were performed using irradiated LDL receptor-deficient (LDLR^−/−) mice with marrow from either TLR4-deficient (TLR4^−/−) or age-matched wild-type (WT) mice. After 12 weeks of being fed a high-cholesterol diet, TLR4^−/− → LDLR^−/− mice developed fewer atherosclerotic lesions in the aorta compared to WT → LDLR^−/− mice. This effect was associated with an increase in multilocular lipid droplets and mitochondria in perivascular adipose tissue (PVAT). Immunofluorescence analysis confirmed that there was an increase in capillary density and M2 macrophage infiltration, accompanied by a decrease in tumour necrosis factor (TNF)-α expression in the localized PVAT of TLR4^−/− → LDLR^−/− mice. In vitro studies indicated that bone marrow-derived macrophages (BMDMs) from WT mice demonstrated an M1-like phenotype and expression of inflammatory cytokines induced by palmitate. These effects were attenuated in BMDMs isolated from TLR4^−/− mice. Furthermore, brown adipocytes incubated with conditioned medium (CM) derived from palmitate-treated BMDMs, exhibited larger and more unilocular lipid droplets, and reduced expression of brown adipocyte-specific markers and perilipin-1 compared to those observed in brown adipocytes exposed to CM from palmitate-treated BMDMs of TLR4^−/− mice. This decreased potency was primarily due to TNF-α, as demonstrated by the capacity of the TNF-α neutralizing antibody to reverse these effects.ConclusionsThese results suggest that haematopoietic-specific deletion of TLR4 promotes PVAT homeostasis, which is involved in reducing macrophage-induced TNF-α secretion and increasing mitochondrial biogenesis in brown adipocytes.     

Dysregulation of the Bmi‐1/p16Ink4a pathway provokes an aging‐associated decline of submandibular gland function

Bmi‐1 prevents stem cell aging, at least partly, by blocking expression of the cyclin‐dependent kinase inhibitor p16^Ink4a. Therefore, dysregulation of the Bmi‐1/p16^Ink4a pathway is considered key to the loss of tissue homeostasis and development of associated degenerative diseases during aging. However, because Bmi‐1 knockout (KO) mice die within 20 weeks after birth, it is difficult to determine exactly where and when dysregulation of the Bmi‐1/p16^Ink4a pathway occurs during aging in vivo. Using real‐time in vivo imaging of p16^Ink4a expression in Bmi‐1‐KO mice, we uncovered a novel function of the Bmi‐1/p16^Ink4a pathway in controlling homeostasis of the submandibular glands (SMGs), which secrete saliva into the oral cavity. This pathway is dysregulated during aging in vivo, leading to induction of p16^Ink4a expression and subsequent declined SMG function. These findings will advance our understanding of the molecular mechanisms underlying the aging‐related decline of SMG function and associated salivary gland hypofunction, which is particularly problematic among the elderly.     

IL-37 inhibits the maturation of dendritic cells through the IL-1R8-TLR4-NF-κB pathway

Mature dendritic cells (DCs) play a pathogenic role in atherosclerosis. Our previous study demonstrated that exogenous interleukin (IL)-37 suppresses the maturation of DCs, induces the T-regulatory (Treg) cell response, and attenuates atherosclerosis in ApoE^−/− mice. The aim of the present study was to explore the molecular mechanism of IL-37 on the maturation of DCs throughout the development of atherosclerosis. The expression of interleukin-1 receptor 8 (IL-1R8), which is a single Ig-domain receptor that was recently found to be pivotal for the extracellular function of IL-37, Toll-like receptor (TLR) 4 and p65, was measured in ApoE^−/− mice and IL-37 transgenic (IL-37tg) ApoE^−/− mice. IL-1R8 was mainly expressed in aortic plaque-infiltrated DCs and at significantly higher levels in IL-37tg atherosclerotic mice, accompanied by lower levels of TLR4 and p65. Furthermore, IL-37 eliminated the maturation of DCs induced by oxidized low-density lipoprotein (oxLDL) and caused marked upregulation of IL-1R8 in vitro and downregulation of TLR4 and p65, which was consistent with the experiments in mice. However, the inhibitory effect of IL-37 on the maturation of DCs in vitro was abolished when IL-37 was used to treat DCs isolated from IL-1R8-deficient and TLR4-deficient mice. Therefore, this study indicated that IL-37 inhibited the maturation of DCs via the IL-1R8-TLR4-NF-κB pathway and attenuated atherosclerosis in ApoE^−/− mice.     

Negative regulation of the hepatic fibrogenic response by suppressor of cytokine signaling 1

Suppressor of cytokine signaling 1 (SOCS1) is an indispensable regulator of IFNγ signaling and has been implicated in the regulation of liver fibrosis. However, it is not known whether SOCS1 mediates its anti-fibrotic functions in the liver directly, or via modulating IFNγ, which has been implicated in attenuating hepatic fibrosis. Additionally, it is possible that SOCS1 controls liver fibrosis by regulating hepatic stellate cells (HSC), a key player in fibrogenic response. While the activation pathways of HSCs have been well characterized, the regulatory mechanisms are not yet clear. The goals of this study were to dissociate IFNγ-dependent and SOCS1-mediated regulation of hepatic fibrogenic response, and to elucidate the regulatory functions of SOCS1 in HSC activation. Liver fibrosis was induced in Socs1^−/−Ifng^−/− mice with dimethylnitrosamine or carbon tetrachloride. Ifng^−/− and C57BL/6 mice served as controls. Following fibrogenic treatments, Socs1^−/−Ifng^−/− mice showed elevated serum ALT levels and increased liver fibrosis compared to Ifng^−/− mice. The latter group showed higher ALT levels and fibrosis than C57BL/6 controls. The livers of SOCS1-deficient mice showed bridging fibrosis, which was associated with increased accumulation of myofibroblasts and abundant collagen deposition. SOCS1-deficient livers showed increased expression of genes coding for smooth muscle actin, collagen, and enzymes involved in remodeling the extracellular matrix, namely matrix metalloproteinases and tissue inhibitor of metalloproteinases. Primary HSCs from SOCS1-deficient mice showed increased proliferation in response to growth factors such as HGF, EGF and PDGF, and the fibrotic livers of SOCS1-deficient mice showed increased expression of the Pdgfb gene. Taken together, these data indicate that SOCS1 controls liver fibrosis independently of IFNγ and that part of this regulation may occur via regulating HSC proliferation and limiting growth factor availability.     

Deletion of p21/Cdkn1a confers protective effect against prostate tumorigenesis in transgenic adenocarcinoma of the mouse prostate model

Cyclin-dependent kinase inhibitors (CDKIs) p21^Cip1/Waf1 (p21) and p27^Kip1 (p27) play a determining role in cell cycle progression by regulating CDK activity; however, p21 role in prostate cancer (PCa) is controversial. Whereas p21 upregulation by anticancer agents causes cell cycle arrest in various PCa cell lines, elevated p21 levels have been associated with higher Gleason score, poor survival and increased PCa recurrence. These conflicting findings suggest that more studies are needed to examine p21 role in PCa. Herein, employing genetic approach, transgenic mice harboring p21/Cdkn1a homozygous deletion (p21^−/−) were crossed with the transgenic adenocarcinoma of the mouse prostate (TRAMP) mice to characterize in vivo consequences of p21 deletion on prostate tumorigenesis. Lower urogenital tract weight of p21^−/−/TRAMP mice was significantly lower than those of p21^+/−/TRAMP and TRAMP mice. Histopathology further supported these observations, showing less aggressiveness in prostates of p21^−/−/TRAMP. Furthermore, a significantly higher incidence of low-grade prostatic intraepithelial lesions (PIN) with a concomitant reduction in adenocarcinoma incidence was observed in p21^−/−/TRAMP mice compared with TRAMP mice. In addition, whereas TRAMP mice showed the presence of poorly differentiated adenocarcinoma lesions, no such lesions were observed in p21/TRAMP transgenic mice. Specifically, there was a significant reduction in the severity of lesions in both p21^−/−/TRAMP and p21^+/−/TRAMP mice compared with TRAMP mice. Together, our data showed that p21 deletion reduces prostate tumorigenesis by slowing-down progression of PIN (pre-malignant) to adenocarcinoma (malignant), suggesting that intact p21 expression is associated with PCa aggressiveness, while its decreased levels may in fact confer protection against prostate tumorigenesis.     

Polycomb Group Protein Bmi1 Is Required for Growth of RAF Driven Non-Small-Cell Lung Cancer

Background

We have previously described a RAF oncogene driven transgenic mouse model for non small cell lung cancer (NSCLC). Here we examine whether tumor initiation and growth requires the stem cell self-renewal factor Bmi1.

Principal Findings

In order to evaluate Bmi1 function in NSCLC two founder lines that differ in incidence and latency of tumor formation were compared. Ablation of Bmi1 expression in both lines had a dramatically decreased tumor growth. As the line with shorter latency matched the life span of Bmi1 knock out mice, these mice were chosen for further study. The absence of Bmi1 did not decrease the number of tumor initiation in these mice as only the size and not the number of tumors decreased. Reduction in tumor growth resulted from an increase in cell death and decrease in cell cycle progression that corresponded with up-regulation of the p16^INK4a and p19^ARF.

Significance

The data identifies Bmi1 as an important factor for expansion but not initiation of RAF driven NSCLC.     

Cover Image,Volume 120, Number 11, November 2019

Skeletal tissue homeostasis is maintained via the balance of osteoclastic bone resorption and osteoblastic bone formation. Autophagy and apoptosis are essential for the maintenance of homeostasis and normal development in cells and tissues. We found that Bax-interacting factor 1 (Bif-1/Endophillin B1/SH3GLB1), involving in autophagy and apoptosis, was upregulated during osteoclastogenesis. Furthermore, mature osteoclasts expressed Bif-1 in the cytosol, particularly the perinuclear regions and podosome, suggesting that Bif-1 regulates osteoclastic bone resorption. Bif-1-deficient (Bif-1 ^−/−) mice showed increased trabecular bone volume and trabecular number. Histological analyses indicated that the osteoclast numbers increased in Bif-1 ^−/− mice. Consistent with the in vivo results, osteoclastogenesis induced by receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL) was accelerated in Bif-1 ^−/− mice without affecting RANKL-induced activation of RANK downstream signals, such as NF-κB and mitogen-activated protein kinases (MAPKs), CD115/RANK expression in osteoclast precursors, osteoclastic bone-resorbing activity and the survival rate. Unexpectedly, both the bone formation rate and osteoblast surface substantially increased in Bif-1 ^−/− mice. Treatment with β-glycerophosphate (β-GP) and ascorbic acid (A.A) enhanced osteoblastic differentiation and mineralization in Bif-1 ^−/− mice. Finally, bone marrow cells from Bif-1 ^−/− mice showed a significantly higher colony-forming efficacy by the treatment with or without β-GP and A.A than cells from wild-type (WT) mice, suggesting that cells from Bif-1 ^−/− mice had higher clonogenicity and self-renewal activity than those from WT mice. In summary, Bif-1 might regulate bone homeostasis by controlling the differentiation and function of both osteoclasts and osteoblasts (235 words).     

Protein deglycase DJ-1 deficiency induces phenotypic switching in vascular smooth muscle cells and exacerbates atherosclerotic plaque instability

Protein deglycase DJ-1 (DJ-1) is a multifunctional protein involved in various biological processes. However, it is unclear whether DJ-1 influences atherosclerosis development and plaque stability. Accordingly, we evaluated the influence of DJ-1 deletion on the progression of atherosclerosis and elucidate the underlying mechanisms. We examine the expression of DJ-1 in atherosclerotic plaques of human and mouse models which showed that DJ-1 expression was significantly decreased in human plaques compared with that in healthy vessels. Consistent with this, the DJ-1 levels were persistently reduced in atherosclerotic lesions of ApoE^−/− mice with the increasing time fed by western diet. Furthermore, exposure of vascular smooth muscle cells (VSMCs) to oxidized low-density lipoprotein down-regulated DJ-1 in vitro. The canonical markers of plaque stability and VSMC phenotypes were evaluated in vivo and in vitro. DJ-1 deficiency in Apoe^−/− mice promoted the progression of atherosclerosis and exaggerated plaque instability. Moreover, isolated VSMCs from Apoe^−/−DJ-1^−/− mice showed lower expression of contractile markers (α-smooth muscle actin and calponin) and higher expression of synthetic indicators (osteopontin, vimentin and tropoelastin) and Kruppel-like factor 4 (KLF4) by comparison with Apoe^−/−DJ-1^+/+ mice. Furthermore, genetic inhibition of KLF4 counteracted the adverse effects of DJ-1 deletion. Therefore, our results showed that DJ-1 deletion caused phenotype switching of VSMCs and exacerbated atherosclerotic plaque instability in a KLF4-dependent manner.     

Apolipoprotein E receptor-2 deficiency enhances macrophage susceptibility to lipid accumulation and cell death to augment atherosclerotic plaque progression and necrosis

Genome-wide association studies have linked LRP8 polymorphisms to premature coronary artery disease and myocardial infarction in humans. However, the mechanisms by which dysfunctions of apolipoprotein E receptor-2 (apoER2), the protein encoded by LRP8 gene, influence atherosclerosis have not been elucidated completely. The current study focused on the role of apoER2 in macrophages, a cell type that plays an important role in atherosclerosis. Results showed that apoER2-deficient mouse macrophages accumulated more lipids and were more susceptible to oxidized LDL (oxLDL)-induced death compared to control cells. Consistent with these findings, apoER2 deficient macrophages also displayed defective serum-induced Akt activation and higher levels of the pro-apoptotic protein phosphorylated p53. Furthermore, the expression and activation of peroxisome proliferator-activated receptor γ (PPARγ) were increased in apoER2-deficient macrophages. Deficiency of apoER2 in hypercholesterolemic LDL receptor-null mice (Lrp8^−/−Ldlr^−/− mice) also resulted in accelerated atherosclerosis with more complex lesions and extensive lesion necrosis compared to Lrp8^+/+Ldlr^−/− mice. The atherosclerotic plaques of Lrp8^−/−Ldlr^−/− mice displayed significantly higher levels of p53-positive macrophages, indicating that the apoER2-deficient macrophages contribute to the accelerated atherosclerotic lesion necrosis observed in these animals. Taken together, this study indicates that apoER2 in macrophages limits PPARγ expression and protects against oxLDL-induced cell death. Thus, abnormal apoER2 functions in macrophages may at least in part contribute to the premature coronary artery disease and myocardial infarction in humans with LRP8 polymorphisms. Moreover, the elevated PPARγ expression in apoER2-deficient macrophages suggests that LRP8 polymorphism may be a genetic modifier of cardiovascular risk with PPARγ therapy.     

Anxiety and increased 5‐HT1A receptor response in NCAM null mutant mice

Mice deficient in the neural cell adhesion molecule (NCAM) show behavioral abnormalities as adults, including altered exploratory behavior, deficits in spatial learning, and increased intermale aggression. Here, we report increased anxiety‐like behavior of homozygous (NCAM^−/−) and heterozygous (NCAM^+/−) mutant mice in a light/dark avoidance test, independent of genetic background and gender. Anxiety‐like behavior was reduced in both NCAM^+/+ and NCAM^−/− mice by systemic administration of the benzodiazepine agonist diazepam and the 5‐HT_1A receptor agonists buspirone and 8‐OH‐DPAT. However, NCAM^−/− mice showed anxiolytic‐like effects at lower doses of buspirone and 8‐OH‐DPAT than NCAM^+/+ mice. Such increased response to 5‐HT_1A receptor stimulation suggests a functional change in the serotonergic system of NCAM^−/− mice, likely involved in the control of anxiety and aggression. However, 5‐HT_1A receptor binding and tissue content of serotonin and its metabolite 5‐hydroxyindolacetic acid were found unaltered in every brain area of NCAM^−/− mice investigated, indicating that expression of 5‐HT_1A receptors as well as synthesis and release of serotonin are largely unchanged in NCAM^−/− mice. We hypothesize a critical involvement of endogenous NCAM in serotonergic transmission via 5‐HT_1A receptors and inwardly rectifying K⁺ channels as the respective effector systems. © 1999 John Wiley & Sons, Inc. J Neurobiol 40: 343–355, 1999     

A high-fat diet temporarily renders Sod1-deficient mice resistant to an oxidative insult

Patients with nonalcoholic fatty liver disease may subsequently develop nonalcoholic steatohepatitis after suffering from a second insult, such as oxidative stress. Aim of this study was to investigate the pathogenesis of the liver injury caused when lipids accumulate under conditions of intrinsic oxidative stress using mice that are deficient in superoxide dismutase 1 (SOD1) and the leptin receptor (Lepr). We established Sod1^−/−::Lepr^db/db mice and carried out analyses of four groups of genetically modified mice, namely, wild type, Sod1^−/−, Lepr^db/db and Sod1^−/−::Lepr^db/db mice. Mice with defects in the SOD1 or Lepr gene are vulnerable to developing fatty livers, even when fed a normal diet. Feeding a high-fat diet (HFD) caused an increase in the number of lipid droplets in the liver to different extents in each genotypic mouse. an HFD caused the accelerated death of db/db mice, but contradictory to our expectations, the death rates for the Sod1-deficient mice were decreased by feeding HFD. Consistent with the improved probability of survival, liver damage was significantly ameliorated by feeding an HFD compared to a normal diet in the mice with an Sod1-deficient background. Oxidative stress markers, hyperoxidized peroxiredoxin and lipid peroxidation products, were decreased somewhat in Sod1^−/− mice by feeding HFD. We conclude that lipids reacted with reactive oxygen species and eliminated them in the livers of the young mice, which resulted in the alleviation of oxidative stress, but in advanced age oxidized products accumulated, leading to the aggravation of the liver injury and an increase in fatality rate.     

Gnpat does not play an essential role in systemic iron homeostasis in murine model

The GNPAT variant rs11558492 (p.D519G) was identified as a novel genetic factor that modifies the iron-overload phenotype in homozygous carriers of the HFE p.C282Y variant. However, the reported effects of the GNPAT p.D519G variant vary among study populations. Here, we investigated the role of GNPAT in iron metabolism using Gnpat-knockout (Gnpat^−/−), Gnpat/Hfe double-knockout (Gnpat^−/−Hfe^−/− or DKO) mice and hepatocyte-specific Gnpat-knockout mice (Gnpat^fl/fl;Alb-Cre). Our analysis revealed no significant difference between wild-type (Gnpat^+/+) and Gnpat^−/− mice, between Hfe^−/− and DKO mice, or between Gnpat^fl/fl and Gnpat^fl/fl;Alb-Cre with respect to serum iron and tissue iron. In addition, the expression of hepcidin was not affected by deleting Gnpat expression in the presence or absence of Hfe. Feeding Gnpat^−/− and DKO mice a high-iron diet had no effect on tissue iron levels compared with wild-type and Hfe^−/− mice, respectively. Gnpat knockdown in primary hepatocytes from wild-type or Hfe^−/− mice did not alter hepcidin expression, but it repressed BMP6-induced hepcidin expression. Taken together, these results support the hypothesis that deleting Gnpat expression has no effect on either systemic iron metabolism or the iron-overload phenotype that develops in Hfe^−/− mice, suggesting that GNPAT does not directly mediate iron homeostasis under normal or high-iron dietary conditions.     

Apaf1 Is Required for Mitochondrial Pathways of Apoptosis and Brain Development

Apoptosis is essential for the precise regulation of cellular homeostasis and development. The role in vivo of Apaf1, a mammalian homolog of C. elegans CED-4, was investigated in gene-targeted Apaf1^−/− mice. Apaf1-deficient mice exhibited reduced apoptosis in the brain and striking craniofacial abnormalities with hyperproliferation of neuronal cells. Apaf1-deficient cells were resistant to a variety of apoptotic stimuli, and the processing of Caspases 2, 3, and 8 was impaired. However, both Apaf1^−/− thymocytes and activated T lymphocytes were sensitive to Fas-induced killing, showing that Fas-mediated apoptosis in these cells is independent of Apaf1. These data indicate that Apaf1 plays a central role in the common events of mitochondria-dependent apoptosis in most death pathways and that this role is critical for normal development.     

Cell cycle arrest and cytochrome c-mediated apoptotic induction by MCS-5A is associated with up-regulation of p16INK4a in HL-60 cells

MCS-5A, an analog of sangivamycin, selectively inhibits the cyclin-dependent kinases CDK1 and 4 in HL-60 cells in vitro (IC₅₀: 9.6 and 8.8 μΜ, respectively), while weakly inhibiting other housekeeping protein kinases. MCS-5A effectively induces HL-60 cell cycle arrest at the G₁ and G₂/M phases through direct inhibition of CDK1 and 4 activity. In addition, elevated expression of p16^INK4a and a reduction in the level of hyperphosphorylated pRb showed that 3 μΜ MCS-5A also induces p16^INK4a-mediated cell cycle arrest at the G₁ phase. Furthermore, apoptotic induction in MCS-5A-treated HL-60 cells is associated with the release of cytochrome c from mitochondria, which, in turn, results in the activation of procaspase-8, -9 and -3, and the cleavage of poly(ADP-ribose) polymerase (PARP). In addition, the involvement of p16^INK4a in this apoptotic induction was demonstrated using A549 cells with a homozygous deletion of p16^INK4a. Based on these results, we conclude that MCS-5A is a candidate therapeutic agent for the treatment of human promyelocytic leukemia via the up-regulation of p16^INK4a.     

Transferrin receptor 2 mitigates periodontitis-driven alveolar bone loss

Periodontitis is associated with significant alveolar bone loss. Patients with iron overload suffer more frequently from periodontitis, however, the underlying mechanisms remain largely elusive. Here, we investigated the role of transferrin receptor 2 (Tfr2), one of the main regulators of iron homeostasis, in the pathogenesis of periodontitis and the dental phenotype under basal conditions in mice. As Tfr2 suppresses osteoclastogenesis, we hypothesized that deficiency of Tfr2 may exacerbate periodontitis-induced bone loss. Mice lacking Tfr2 (Tfr2^−/−) and wild-type (Tfr2^+/+) littermates were challenged with experimental periodontitis. Mandibles and maxillae were collected for microcomputed tomography and histology analyses. Osteoclast cultures from Tfr2^+/+ and Tfr2^−/− mice were established and analyzed for differentiation efficiency, by performing messenger RNA expression and protein signaling pathways. After 8 days, Tfr2-deficient mice revealed a more severe course of periodontitis paralleled by higher immune cell infiltration and a higher histological inflammation index than Tfr2^+/+ mice. Moreover, Tfr2-deficient mice lost more alveolar bone compared to Tfr2^+/+ littermates, an effect that was only partially iron-dependent. Histological analysis revealed a higher number of osteoclasts in the alveolar bone of Tfr2-deficient mice. In line, Tfr2-deficient osteoclastic differentiation ex vivo was faster and more efficient as reflected by a higher number of osteoclasts, a higher expression of osteoclast markers, and an increased resorptive activity. Mechanistically, Tfr2-deficient osteoclasts showed a higher p38-MAPK signaling and inhibition of p38-MAPK signaling in Tfr2-deficient cells reverted osteoclast formation to Tfr2^+/+ levels. Taken together, our data indicate that Tfr2 modulates the inflammatory response in periodontitis thereby mitigating effects on alveolar bone loss.     

Loss of CDKN2A expression is a frequent event in primary invasive melanoma and correlates with sensitivity to the CDK4/6 inhibitor PD0332991 in melanoma cell lines

We have investigated the potential for the p16‐cyclin D‐CDK4/6‐retinoblastoma protein pathway to be exploited as a therapeutic target in melanoma. In a cohort of 143 patients with primary invasive melanoma, we used fluorescence in situ hybridization to detect gene copy number variations (CNVs) in CDK4, CCND1, and CDKN2A and immunohistochemistry to determine protein expression. CNVs were common in melanoma, with gain of CDK4 or CCND1 in 37 and 18% of cases, respectively, and hemizygous or homozygous loss of CDKN2A in 56%. Three‐quarters of all patients demonstrated a CNV in at least one of the three genes. The combination of CCND1 gain with either a gain of CDK4 and/or loss of CDKN2A was associated with poorer melanoma‐specific survival. In 47 melanoma cell lines homozygous loss, methylation or mutation of CDKN2A gene or loss of protein (p16^INK4A) predicted sensitivity to the CDK4/6 inhibitor PD0332991, while RB1 loss predicted resistance.     

Cardiovascular effects of exogenous adrenomedullin and CGRP in Ramp and Calcrl deficient mice

Adrenomedullin (AM) and calcitonin gene-related peptide (CGRP) are potent vasodilator peptides and serve as ligands for the G-protein coupled receptor (GPCR) calcitonin receptor-like receptor (CLR/Calcrl). Three GPCR accessory proteins called receptor activity-modifying proteins (RAMPs) modify the ligand binding affinity of the receptor such that the CLR/RAMP1 heterodimer preferably binds CGRP, while CLR/RAMP2 and CLR/RAMP3 have a stronger affinity for AM. Here we determine the contribution of each of the three RAMPs to blood pressure control in response to exogenous AM and CGRP by measuring the blood pressure of mice with genetic reduction or deletion of the receptor components. Thus, the cardiovascular response of Ramp1^−/−, Ramp2^+/−, Ramp3^−/−, Ramp1^−/−/Ramp3^−/− double-knockout (dKO), and Calcrl^+/− mice to AM and CGRP were compared to wildtype mice. While under anesthesia, Ramp1^−/− male mice had significantly higher basal blood pressure than wildtype males; a difference which was not present in female mice. Additionally, anesthetized Ramp1^−/−, Ramp3^−/−, and Calcrl^+/− male mice exhibited significantly higher basal blood pressure than females of the same genotype. The hypotensive response to intravenously injected AM was greatly attenuated in Ramp1^−/− mice, and to a lesser extent in Ramp3^−/− and Calcrl^+/− mice. However, Ramp1^−/−/Ramp3^−/− dKO mice retained some hypotensive response to AM. These results suggest that the hypotensive effect of AM is primarily mediated through the CLR/RAMP1 heterodimer, but that AM signaling via CLR/RAMP2 and CLR/RAMP3 also contributes to some hypotensive action. On the other hand, CGRP’s hypotensive activity seems to be predominantly through the CLR/RAMP1 heterodimer. With this knowledge, therapeutic AM or CGRP peptides could be designed to cause less hypotension while maintaining canonical receptor-RAMP mediated signaling.