The effects of preosteoblast-derived exosomes on macrophages and bone in mice.

The effect of preosteoblast-derived exosomes on bone marrow macrophages (BMMΦ) and calvarial osteoblasts (cOB) was evaluated in vitro, and bone formation studies were performed in vivo in mice. Preosteoblastic MC3T3-E1 clone 4 (MC4) cell-derived exosomes (MC4exo) were characterized with particle tracking, transmission electron microscopy and western blot analysis to validate size, number, shape and phenotypic exosome markers. Exosomes pre-labelled with PKH67 were incubated with BMMΦ and phagocytosis of exosomes was confirmed. To examine the effect of MC4exo on macrophage polarization, BMMΦ were treated with MC4exo and the expression of pro- and anti-inflammatory cytokines was determined by qPCR. MC4exo treatment upregulated mRNA expression of Cd86, Il1ß, Ccl2, Rankl and Nos, and downregulated Cd206, Il10 and Tnfα, suggesting a shift towards pro-inflammatory 'M1-like' macrophage polarization. Combination of RANKL and MC4exo increased osteoclast differentiation of BMMΦ in comparison to RANKL alone as analysed by TRAP staining. MC4exo treatment showed no significant effect on calvarial osteoblast mineralization. For in vivo studies, intratibial inoculation of MC4exo (2 × 109 particles in PBS, n = 12) and vehicle control (PBS only, n = 12) was performed in C57Bl/6 mice (8 weeks, male). Micro-CT analyses of the trabecular and cortical bone compartments were assessed at 4 weeks post-injection. Tibial sections were stained for TRAP activity to determine osteoclast presence and immunofluorescence staining was performed to detect osteocalcin (Ocn), osterix (Osx) and F4/80 expression. Intratibial inoculation of MC4exo increased the diaphyseal bone mineral density and trabecular bone volume fraction due to increased trabecular number. This increase in bone was accompanied by a reduction in bone marrow macrophages and osteoclasts at the experimental endpoint. Together, these findings suggest that preosteoblast-derived exosomes enhanced bone formation by influencing macrophage responses.

Bone Marrow Macrophages Induce Inflammation by Efferocytosis of Apoptotic Prostate Cancer Cells via HIF-1α Stabilization.

The clearance of apoptotic cancer cells by macrophages, known as efferocytosis, fuels the bone-metastatic growth of prostate cancer cells via pro-inflammatory and immunosuppressive processes. However, the exact molecular mechanisms remain unclear. In this study, single-cell transcriptomics of bone marrow (BM) macrophages undergoing efferocytosis of apoptotic prostate cancer cells revealed a significant enrichment in their cellular response to hypoxia. Here, we show that BM macrophage efferocytosis increased hypoxia inducible factor-1alpha (HIF-1α) and STAT3 phosphorylation (p-STAT3 at Tyr705) under normoxic conditions, while inhibitors of p-STAT3 reduced HIF-1α. Efferocytosis promoted HIF-1α stabilization, reduced its ubiquitination, and induced HIF-1α and p-STAT3 nuclear translocation. HIF-1α stabilization in efferocytic BM macrophages resulted in enhanced expression of pro-inflammatory cytokine MIF, whereas BM macrophages with inactive HIF-1α reduced MIF expression upon efferocytosis. Stabilization of HIF-1α using the HIF-prolyl-hydroxylase inhibitor, Roxadustat, enhanced MIF expression in BM macrophages. Furthermore, BM macrophages treated with recombinant MIF protein activated NF-κB (p65) signaling and increased the expression of pro-inflammatory cytokines. Altogether, these findings suggest that the clearance of apoptotic cancer cells by BM macrophages triggers p-STAT3/HIF-1α/MIF signaling to promote further inflammation in the bone tumor microenvironment where a significant number of apoptotic cancer cells are present.

Contribution of Macrophages and T Cells in Skeletal Metastasis.

Bone is a common site for metastases with a local microenvironment that is highly conducive for tumor establishment and growth. The bone marrow is replete with myeloid and lymphoid linage cells that provide a fertile niche for metastatic cancer cells promoting their survival and growth. Here, we discuss the role of macrophages and T cells in pro- and anti-tumoral mechanisms, their interaction to support cancer cell growth, and their contribution to the development of skeletal metastases. Importantly, immunotherapeutic strategies targeting macrophages and T cells in cancer are also discussed in this review as they represent a great promise for patients suffering from incurable bone metastases.

Unique Pro-Inflammatory Response of Macrophages during Apoptotic Cancer Cell Clearance.

The clearance of apoptotic cells by macrophages (efferocytosis) is crucial to maintain normal tissue homeostasis; however, efferocytosis of cancer cells frequently results in inflammation and immunosuppression. Recently, we demonstrated that efferocytosis of apoptotic prostate cancer cells by bone marrow-derived macrophages induced a pro-inflammatory response that accelerated metastatic tumor growth in bone. To evaluate the microenvironmental impact of macrophages and their efferocytic function, we compared peritoneal macrophages (P-MΦ) versus bone marrow-derived macrophages (BM-MΦs) using an efferocytosis in vitro model. The capability to engulf apoptotic prostate cells was similar in BM-MΦs and P-MΦs. Ex vivo analysis of BM-MΦs showed an M2-like phenotype compared with a predominantly M1-like phenotype in P-MΦs. A distinct gene and protein expression profile of pro-inflammatory cytokines was found in BM-MΦs as compared with P-MΦs engulfing apoptotic prostate cancer cells. Importantly, the reprogramming of BM-MΦs toward an M1-like phenotype mitigated their inflammatory cytokine expression profile. In conclusion, BM-MΦs and P-MΦs are both capable of efferocytosing apoptotic prostate cancer cells; however, BM-MΦs exert increased inflammatory cytokine expression that is dependent upon the M2 polarization stage of macrophages. These findings suggest that bone marrow macrophage efferocytosis of apoptotic cancer cells maintains a unique pro-inflammatory microenvironment that may support a fertile niche for cancer growth. Finally, bone marrow macrophage reprogramming towards M1-type by interferon-γ (IFN-γ) induced a significant reduction in the efferocytosis-mediated pro-inflammatory signature.

Plau and Tgfbr3 are YAP-regulated genes that promote keratinocyte proliferation.

Yes-associated protein (YAP) is a mechanosensor protein and a downstream effector of the Hippo kinase pathway, which controls organ growth, cell proliferation, survival, maintenance and regeneration. Unphosphorylated YAP translocates to the nucleus where it acts as a cofactor of primarily the TEAD transcription factors to activate target gene transcription and cell proliferation. Perturbed YAP activation results in tumorigenesis. The pathways downstream of activated YAP that drive cell proliferation remain relatively unexplored. In this study, we employed YAP2-5SA-∆C transgenic mice, which overexpress a mildly activated YAP mutant protein in basal keratinocytes leading to increased proliferation of the epidermal stem/progenitor cell populations. We performed massively-parallel sequencing of skin biopsy mRNA (RNA-Seq) and found dysregulation of 1491 genes in YAP2-5SA-∆C skin, including many with roles in cell activation and proliferation. Furthermore, we found that 150 of these dysregulated genes harbored YAP/TEAD binding motifs in the 3' UTR, suggesting that these may be direct YAP/TEAD target genes in the control of epidermal regeneration. Further validation and functional characterization assays identified Plau and Tgfbr3 as prime candidate genes that may be activated by epidermal YAP activity in the mouse skin in vivo to promote keratinocyte proliferation. This study provides novel insights into the mechanisms regulated by YAP that control tissue homeostasis, and in particular in conditions where YAP is aberrantly activated such as in neoplastic and regenerative skin disease.

Epidermal YAP activity drives canonical WNT16/ß-catenin signaling to promote keratinocyte proliferation in vitro and in the murine skin.

The skin constantly self-renews throughout adult life. Wnt/ß-catenin signaling plays a key role in promoting keratinocyte proliferation in the hair follicles and in the interfollicular epidermis. A recent report demonstrated that epidermal YAP activity drives ß-catenin activation to promote keratinocyte proliferation in the murine skin. However, it remains unclear whether this is caused by paracrine activation of canonical Wnt signaling or through other YAP/ß-catenin regulatory interactions. In the present study, we found that XAV939-inhibition of canonical WNT signaling in skin of YAP2-5SA-ΔC mice resulted in diminished ß-catenin activation, reduced keratinocyte proliferation, and a mitigation of the hyperplastic abnormalities in the interfollicular epidermis, signifying a canonical WNT ligand-dependent mechanism. Our subsequent analyses determined that WNT16 is produced in response to YAP activity in keratinocytes both in vitro and in vivo, and that WNT16 drives HaCaT keratinocyte proliferation via canonical WNT16/ß-catenin signaling. We conclude that under normal physiological conditions WNT16 is the paracrine WNT ligand secreted in response to epidermal YAP activity that promotes cell proliferation in the interfollicular epidermis. This study delineates a fundamental YAP-driven mechanism that controls normal skin regeneration, and that may be perturbed in human regenerative disease displaying increased YAP and WNT signaling activity.

Positive regulatory interactions between YAP and Hedgehog signalling in skin homeostasis and BCC development in mouse skin in vivo.

Skin is a highly plastic tissue that undergoes tissue turnover throughout life, but also in response to injury. YAP and Hedgehog signalling play a central role in the control of epidermal stem/progenitor cells in the skin during embryonic development, in postnatal tissue homeostasis and in skin carcinogenesis. However, the genetic contexts in which they act to control tissue homeostasis remain mostly unresolved. We provide compelling evidence that epidermal YAP and Hedgehog/GLI2 signalling undergo positive regulatory interactions in the control of normal epidermal homeostasis and in basal cell carcinoma (BCC) development, which in the large majority of cases is caused by aberrant Hedgehog signalling activity. We report increased nuclear YAP and GLI2 activity in the epidermis and BCCs of K14-CreER/Rosa-SmoM2 transgenic mouse skin, accompanied with increased ROCK signalling and ECM remodelling. Furthermore, we found that epidermal YAP activity drives GLI2 nuclear accumulation in the skin of YAP2-5SA-ΔC mice, which depends on epidermal ß-catenin activation. Lastly, we found prominent nuclear activity of GLI2, YAP and ß-catenin, concomitant with increased ROCK signalling and stromal fibrosis in human BCC. Our work provides novel insights into the molecular mechanisms underlying the interplay between cell signalling events and mechanical force in normal tissue homeostasis in vivo, that could potentially be perturbed in BCC development.

Pyrimidine tract-binding protein 1 mediates pyruvate kinase M2-dependent phosphorylation of signal transducer and activator of transcription 3 and oncogenesis in anaplastic large cell lymphoma.

PKM2 (pyruvate kinase M2), a critical regulator of glycolysis, is phosphorylated by numerous growth factor receptors and oncogenic tyrosine kinases including NPM-ALK which is expressed in a subset of aggressive T-cell non-Hodgkin lymphomas known as anaplastic large cell lymphoma, ALK-positive. Our previous work demonstrated that phosphorylation of Y105-PKM2 by NPM-ALK regulates a major metabolic shift to promote lymphomagenesis. In addition to its role in metabolism, recent studies have shown that PKM2 promotes oncogenesis by phosphorylating nuclear STAT3 (signal transducer and activator of transcription 3) and regulating transcription of genes involved in cell survival and proliferation. We hypothesized that identification of novel PKM2 interactors could provide additional insights into its expanding functional role in cancer. To this end, immunocomplexes of FLAG-tagged PKM2 were isolated from NPM-ALK-positive ALCL (anaplastic large cell lymphoma) cells and subjected to liquid chromatography tandem mass spectrometry (LC-MS/MS) which led to the identification of polypyrimidine tract-binding protein (PTBP1) as a novel interactor of PKM2. The interaction between PTBP1 and PKM2 was restricted to the nucleus and was dependent on NPM-ALK mediated Y105 phosphorylation of PKM2. Stable shRNA-mediated silencing of PTBP1 resulted in a marked decrease in pY105-PKM2 and pY705-STAT3 which led to decreased ALCL cell proliferation and colony formation. Overall, our data demonstrate that PTBP1 interacts with PKM2 and promotes ALCL oncogenesis by facilitating PKM2-dependent activation of STAT3 within the nucleus.

Epidermal YAP2-5SA-ΔC Drives ß-Catenin Activation to Promote Keratinocyte Proliferation in Mouse Skin In Vivo.

The epidermis is a highly regenerative tissue. YAP is a pivotal regulator of stem/progenitor cells in tissue regeneration, including in the epidermis. The molecular mechanisms downstream of YAP that activate epidermal cell proliferation remain largely unknown. We found that YAP and ß-catenin co-localize in the nuclei of keratinocytes in the regenerating epidermis in vivo and in proliferating HaCaT keratinocytes in vitro. Inactivation of YAP in HaCaT keratinocytes resulted in reduced activated ß-catenin and reduced keratinocyte numbers in vitro. In addition, we found that in the hyperplastic epidermis of YAP2-5SA-ΔC mice, the mutant YAP2-5SA-ΔC protein was predominantly localized in the keratinocyte nuclei and caused increased expression of activated nuclear ß-catenin. Accordingly, ß-catenin transcriptional activity was elevated in the skin of live YAP2-5SA-ΔC/TOPFLASH mice. Lastly, loss of ß-catenin in basal keratinocytes of YAP2-5SA-ΔC/K14-creERT/CtnnB1-/- mice resulted in reduced proliferation of basal keratinocytes and a striking rescue of the hyperplastic abnormalities. Taken together, our work shows that YAP2-5SA-ΔC drives ß-catenin activity to promote basal keratinocyte proliferation in the mouse skin in vivo. Our data shine new light on the etiology of regenerative dermatological disorders and other human diseases that display increased YAP and ß-catenin activity.

Novel and known MYOC exon 3 mutations in an admixed Peruvian primary open-angle glaucoma population.

PURPOSE: The aim of this study was to characterize a representative sample of the Peruvian population suffering open-angle glaucoma (OAG) with respect to the myocilin gene (MYOC) mutations, glaucoma phenotype, and ancestry for future glaucoma risk assessment. METHODS: DNA samples from 414 unrelated Peruvian subjects, including 205 open-angle glaucoma cases (10 juvenile glaucoma [JOAG], 19 normal-tension glaucoma [NTG], and 176 POAG) and 209 randomly sampled controls, were screened for nucleotide changes in MYOC exon 3 by conformational sensitive gel electrophoresis (CSGE) and mutation screening. RESULTS: We identified a probable causative novel MYOC missense mutation, Gly326Ser, in one POAG case and found a consistent genotype-phenotype correlation in eight of his relatives. We also found the known causative MYOC mutation Trp286Arg in one JOAG case and one POAG case. A known causative single base MYOC deletion, T1357, was found in one POAG case. Two previously reported silent polymorphisms, Thr325Thr and Tyr347Tyr, were found in both the case and the control populations. A novel missense variant, Met476Arg, was identified in two unrelated controls. CONCLUSIONS: The screening of exon 3 of MYOC in a representative sample of 205 independent POAG patients from Peru and 209 matched controls identified novel and previously reported mutations (both pathogenic and nonpathogenic) from other global regions. These results reflect the complex admixture of Amerindian and Old World ancestry in urban populations of Latin America, in general, and in Peru, in particular. It will be important to gather information about the ancestral origin of MYOC and other POAG gene mutations to develop screening panels and risk assessment for POAG in Peru.