Macrophages are required for tissue homeostasis through their role in regulation of the immune response and the resolution of injury. Here we show, using the kidney as a model, that the Wnt pathway ...ligand Wnt7b is produced by macrophages to stimulate repair and regeneration. When macrophages are inducibly ablated from the injured kidney, the canonical Wnt pathway response in kidney epithelial cells is reduced. Furthermore, when Wnt7b is somatically deleted in macrophages, repair of injury is greatly diminished. Finally, injection of the Wnt pathway regulator Dkk2 enhances the repair process and suggests a therapeutic option. Because Wnt7b is known to stimulate epithelial responses during kidney development, these findings suggest that macrophages are able to rapidly invade an injured tissue and reestablish a developmental program that is beneficial for repair and regeneration.
When and where to make or break new blood vessel connections is the key to understanding guided vascular patterning. VEGF-A stimulation and Dll4/Notch signaling cooperatively control the number of ...new connections by regulating endothelial tip cell formation. Here, we show that the Notch-regulated ankyrin repeat protein (Nrarp) acts as a molecular link between Notch- and Lef1-dependent Wnt signaling in endothelial cells to control stability of new vessel connections in mouse and zebrafish. Dll4/Notch-induced expression of Nrarp limits Notch signaling and promotes Wnt/Ctnnb1 signaling in endothelial stalk cells through interactions with Lef1. BATgal-reporter expression confirms Wnt signaling activity in endothelial stalk cells. Ex vivo, combined Wnt3a and Dll4 stimulation of endothelial cells enhances Wnt-reporter activity, which is abrogated by loss of Nrarp. In vivo, loss of Nrarp, Lef1, or endothelial Ctnnb1 causes vessel regression. We suggest that the balance between Notch and Wnt signaling determines whether to make or break new vessel connections.
Myeloid cells are a feature of most tissues. Here we show that during development, retinal myeloid cells (RMCs) produce Wnt ligands to regulate blood vessel branching. In the mouse retina, where ...angiogenesis occurs postnatally, somatic deletion in RMCs of the Wnt ligand transporter Wntless results in increased angiogenesis in the deeper layers. We also show that mutation of Wnt5a and Wnt11 results in increased angiogenesis and that these ligands elicit RMC responses via a non-canonical Wnt pathway. Using cultured myeloid-like cells and RMC somatic deletion of Flt1, we show that an effector of Wnt-dependent suppression of angiogenesis by RMCs is Flt1, a naturally occurring inhibitor of vascular endothelial growth factor (VEGF). These findings indicate that resident myeloid cells can use a non-canonical, Wnt-Flt1 pathway to suppress angiogenic branching.
Circadian clocks regulate various aspects of photoreceptor physiology, but their contribution to photoreceptor development and function is unclear. Cone photoreceptors are critical for color vision. ...Here, we define the molecular function of circadian activity within cone photoreceptors and reveal a role for the clock genes Bmal1 and Per2 in regulating cone spectral identity. ChIP analysis revealed that BMAL1 binds to the promoter region of the thyroid hormone (TH)-activating enzyme type 2 iodothyronine deiodinase (Dio2) and thus regulates the expression of Dio2. TH treatment resulted in a partial rescue of the phenotype caused by the loss of Bmal1, thus revealing a functional relationship between Bmal1 and Dio2 in establishing cone photoreceptor identity. Furthermore, Bmal1 and Dio2 are required to maintain cone photoreceptor functional integrity. Overall, our results suggest a mechanism by which circadian proteins can locally regulate the availability of TH and influence tissue development and function.
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•Circadian clock genes Bmal1 and Per2 are required for spatial patterning of cone opsins•BMAL1 controls expression of the thyroid hormone-activating gene Dio2•Regulation of Dio2 by BMAL1 is required for cone spectral identity•Bmal1 and Dio2 are required for visual function and cone integrity
Short- and medium-wavelength opsins in cone photoreceptors are required for color vision. Sawant et al. show that core circadian clock genes are required for cone opsin expression, gradient maintenance, and cone function and that these effects are mediated by regulation of thyroid hormone signaling.
Recent genome-wide association studies of individuals of Asian and European descent have found that SNPs located within the genomic region (1p31.3) encoding the Wntless (Wls)/Gpr177 protein are ...associated significantly with reduced bone mineral density. Wls / Gpr177 is a newly identified chaperone protein that specifically escorts Wnt ligands for secretion. Given the strong functional association between the Wnt signaling pathways and bone development and homeostasis, we generated osteoblast-specific Wls-deficient (Ocn-Cre;Wls-flox) mice. Homozygous conditional knockout animals were born at a normal Mendelian frequency. Whole-body dual-energy X-ray absorptiometry scanning revealed that bone-mass accrual was significantly inhibited in homozygotes as early as 20 d of age. These homozygotes had spontaneous fractures and a high frequency of premature lethality at around 2 mo of age. Microcomputed tomography analysis and histomorphometric data revealed a dramatic reduction of both trabecular and cortical bone mass in homozygous mutants. Bone formation in homozygotes was severely impaired, but no obvious phenotypic change was observed in mice heterozygous for the conditional deletion. In vitro studies showed that Wls -deficient osteoblasts had a defect in differentiation and mineralization, with significant reductions in the expression of key osteoblast differentiation regulators. In summary, these results reveal a surprising and crucial role of osteoblast-secreted Wnt ligands in bone-mass accrual.
Almost all life forms can detect and decode light information for adaptive advantage. Examples include the visual system, in which photoreceptor signals are processed into virtual images, and the ...circadian system, in which light entrains a physiological clock. Here we describe a light response pathway in mice that employs encephalopsin (OPN3, a 480 nm, blue-light-responsive opsin) to regulate the function of adipocytes. Germline null and adipocyte-specific conditional null mice show a light- and Opn3-dependent deficit in thermogenesis and become hypothermic upon cold exposure. We show that stimulating mouse adipocytes with blue light enhances the lipolysis response and, in particular, phosphorylation of hormone-sensitive lipase. This response is Opn3 dependent. These data establish a key mechanism in which light-dependent, local regulation of the lipolysis response in white adipocytes regulates energy metabolism.
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•Adipocytes express encephalopsin (OPN3), a 480 nm blue-light-sensitive opsin•Mice lacking OPN3 or blue light have diminished thermogenesis during cold exposure•Loss of OPN3 reduces oxygen consumption and energy expenditure•White adipocyte OPN3 promotes lipolysis during cold exposure
White adipocytes activate the lipolysis pathway to produce the free fatty acids that are used as heating fuel by brown adipose tissue. Nayak et al. show that Opsin 3 is required for blue-light-enhanced activation of the lipolysis pathway. This explains the low body temperature of Opn3 mutant mice.
Dishevelled (Dvl) is an essential protein in the Wnt signaling pathways; it uses its PDZ domain to transduce the Wnt signals from the membrane receptor Frizzled to downstream components. Here, we ...report identifying a drug-like small molecule compound through structure-based ligand screening and NMR spectroscopy and show the compound to interact at low micromolar affinity with the PDZ domain of Dvl. In a Xenopus testing system, the compound could permeate the cell membrane and block the Wnt signaling pathways. In addition, the compound inhibited Wnt signaling and reduced the levels of apoptosis in the hyaloid vessels of eye. Moreover, this compound also suppressed the growth of prostate cancer PC-3 cells. These biological effects suggest that by blocking the PDZ domain of Dvl, the compound identified in our studies effectively inhibits the Wnt signaling and thus provides a useful tool for studies dissecting the Wnt signaling pathways.
Macrophages have a critical role in inflammatory and immune responses through their ability to recognize and engulf apoptotic cells. Here we show that macrophages initiate a cell-death programme in ...target cells by activating the canonical WNT pathway. We show in mice that macrophage WNT7b is a short-range paracrine signal required for WNT-pathway responses and programmed cell death in the vascular endothelial cells of the temporary hyaloid vessels of the developing eye. These findings indicate that macrophages can use WNT ligands to influence cell-fate decisions-including cell death-in adjacent cells, and raise the possibility that they do so in many different cellular contexts.
BackgroundThe cGAS-STING pathway is a key mediator in boosting anti-tumor responses through activation of the type I interferon signaling cascade. Major limitations of STING agonists currently ...studied include poor pharmacokinetic and physicochemical properties, and risks of adverse effects due to induction of excessive cytokines following systemic administration. An alternative STING activation strategy, that may improve efficacy and safety, is to inhibit the extracellular hydrolase, ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), a negative regulator of the STING pathway, which directly hydrolyses 2′3′-cGAMP, the natural ligand for STING. Increased ENPP1 expression has been shown to be associated with reduced immune cell infiltration and poor prognosis in multiple tumor types. Inhibition of ENPP1 would prevent abolishment of the cGAS-STING-mediated immune activation, and allow for modulation of the tumor microenvironment with more T-cell infiltration and dendritic cell activation. Here, we report ISM5939 is a novel, potent, selective inhibitor of ENPP1 with oral bioavailability.MethodsISM5939 was designed with the assistance of Chemistry42, an AI platform. An ENPP1 enzyme inhibitory activity assay was used to assess the bioactivity of ISM5939. Cellular ENPP1 inhibitory activity was assessed using a 2’,3’-cGAMP ELISA assay in the triple negative breast cancer cell line, MDA-MB-231. In vivo studies were conducted in the MC-38 and CT-26 syngeneic colorectal carcinoma mouse models.ResultsISM5939 potently inhibited ENPP1 with an IC50 of 0.63 nM in the enzymatic assay, and an EC50 of 330 nM in the MDA-MB-231 cellular assay. ISM5939 monotherapy (30 mg/kg, p.o. BID) demonstrated a 67% Tumor Growth Inhibition (TGI) in the MC38 model. Further, ISM5939 dose-dependently enhanced the efficacy of anti-PD-L1 or anti-PD-1 therapy. At 30 mg/kg, p.o. BID, ISM5939 in combination with anti-PD-L1 or anti-PD-1 exhibited synergistic effects, with TGI of 96% vs 53% with anti-PD-L1 alone, and 68% vs 28% with anti-PD-1 antibody alone. Furthermore, ISM5939 administration did not result in a significant change in body weight.ISM5939 displayed favorable ADME properties. At 10 uM, ISM5939 showed no obvious agonistic or antagonistic effects on 44 selected targets in a mini safety panel, demonstrating its selectivity as an ENPP1 inhibitor. Further, results from a 28-day rat and dog DRF study revealed a good safety margin for ISM5939.ConclusionsISM5939, is a novel, potent ENPP1 inhibitor that augments the effectiveness of anti-PD-L1 or anti-PD1 therapy in multiple syngeneic mouse tumor models, while exhibiting favorable safety and tolerability properties. Together, these data support further evaluation of ISM5939 as an anti-tumor agent.
Ambient light is both a stimulus for visual function and a regulator of photoreceptor physiology. However, it is not known if light can regulate any aspect of photoreceptor development. The purpose ...of this study was to investigate whether ambient light is required for the development of mouse rod photoreceptors.
Newborn mouse pups (C57BL/6) were reared in either cyclic light (LD) or constant dark (DD). Pups were collected at postnatal day (P)5, P10, P17, or P24. We performed retinal morphometric and cell death analysis at P5, P10, and P17. Rhodopsin expression was assessed using immunofluorescence, Western blot, and quantitative RT-PCR analysis. Electroretinograms were performed at P17 and P24. Radioimmunoassay and ELISA were used to follow changes in thyroid hormone levels in the serum and vitreous.
In the DD pups, the outer nuclear layer was significantly thinner at P10 and there were higher numbers of apoptotic cells at P5 compared to the LD pups. Rhodopsin expression was lower at P10 and P17 in DD pups. Electroretinogram a-waves were reduced in amplitude at P17 in the DD pups. The DD animals had lower levels of circulating thyroid hormones at P10. Light-mediated changes in thyroid hormones occur as early as P5, as we detected lower levels of total triiodothyronine in the vitreous from the DD animals. Drug-induced developmental hypothyroidism resulted in lower rhodopsin expression at P10.
Our data demonstrate that light exposure during postnatal development is required for rod photoreceptor development and that this effect could be mediated by thyroid hormone signaling.
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