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.

Efferocytosis and Bone Dynamics.

PURPOSE OF REVIEW: This review summarizes the recently published scientific evidence regarding the role of efferocytosis in bone dynamics and skeletal health. RECENT FINDINGS: Several types of efferocytes have been identified within the skeleton, with macrophages being the most extensively studied. Efferocytosis is not merely a 'clean-up' process vital for maintaining skeletal homeostasis; it also plays a crucial role in promoting resolution pathways and orchestrating bone dynamics, such as osteoblast-osteoclast coupling during bone remodeling. Impaired efferocytosis has been associated with aging-related bone loss and various skeletal pathologies, including osteoporosis, osteoarthritis, rheumatoid arthritis, and metastatic bone diseases. Accordingly, emerging evidence suggests that targeting efferocytic mechanisms has the potential to alleviate these conditions. While efferocytosis remains underexplored in the skeleton, recent discoveries have shed light on its pivotal role in bone dynamics, with important implications for skeletal health and pathology. However, there are several knowledge gaps and persisting technical limitations that must be addressed to fully unveil the contributions of efferocytosis in bone.

Evaluating Factors Associated With Patient-Perceived Applicability of the QuickDASH Survey in Reflecting Their Goals of Care Prior to Hand Surgery.

PURPOSE: Patient-reported outcome measures intend to capture patients' perspectives on their health status. However, the patient-perceived applicability of many of these patient-reported outcome measures is unknown. We hypothesized that patients experiencing greater upper extremity disability and greater pain interference would be more likely to report that the Quick Disabilities of the Arm, Shoulder, and Hand questionnaire (QuickDASH) survey content is responsive to their daily lives and goals in seeking surgical care. METHODS: Adult preoperative hand surgery patients at a single tertiary academic center were recruited prospectively. QuickDASH, Patient-Reported Outcomes Measurement Information System Pain Interference computerized-adaptive-testing, and the Godin Leisure-Time Exercise Questionnaire (GLTEQ)-a validated adult physical activity level metric-data were collected. The following two Likert response questions were also asked: question (1) "How applicable is the above questionnaire to your treatment goals for your upper extremity condition?" and question (2) "How applicable is the overall questionnaire to your daily life?" Multivariable binary logistic regression was performed to define the factors associated with patients reporting that the survey was "very applicable." RESULTS: Of the 133 included patients, the mean age was 49 ± 18 years, 40% were women, and the mean GLTEQ score was 54.1 ± 5.5 (consistent with a high level of activity). For questions 1 and 2, 32% and 29% of the patients reported that QuickDASH was "very applicable," respectively. The multivariable model demonstrated that for every 10-point increase in QuickDASH, there was a 45% to 49% greater odds of respondents reporting that the survey was "very applicable," and for every 5-point increase in pain interference computerized-adaptive-testing, the odds increased by 55% to 70%. No association with GLTEQ was observed. CONCLUSIONS: Patients with greater upper extremity disability and pain interference were more likely to find the QuickDASH content to be applicable to their daily lives and goals in seeking surgical care. CLINICAL RELEVANCE: These findings suggest that QuickDASH may not be an optimal instrument when evaluating upper extremity function in cohorts with mild disability and low pain interference.

Inflammatory bone loss associated with MFG-E8 deficiency is rescued by teriparatide.

A prolonged increase in proinflammatory cytokines is associated with osteoporotic and autoimmune bone loss and, conversely, anti-inflammatory pathways are associated with protection against bone loss. Milk fat globule-epidermal growth factor (MFG-E)-8 is a glycoprotein that is proresolving, regulates apoptotic cell clearance, and has been linked to autoimmune disease and skeletal homeostasis. The role of MFG-E8 in the young vs. adult skeleton was determined in mice deficient in MFG-E8 (KO). In vivo, trabecular bone was similar in MFG-E8KO and wild-type (WT) mice at 6 and 16 wk, whereas 22 wk adult MFG-E8KO mice displayed significantly reduced trabecular BV/TV. The number of osteoclasts per bone surface was increased in 22-wk MFG-E8 KO vs. WT mice, and recombinant murine MFG-E8 decreased the number and size of osteoclasts in vitro. Adult MFG-E8KO spleen weight:body weight was increased compared with WT, and flow cytometric analysis showed significantly increased myeloid-derived suppressor cells (CD11bhiGR-1+) and neutrophils (CD11bhiLy6G+) in MFG-E8KO bone marrow, suggesting an inflammatory phenotype. PTH-treated MFG-E8KO mice showed a greater anabolic response (+124% BV/TV) than observed in PTH-treated WT mice (+64% BV/TV). These data give insight into the role of MFG-E8 in the adult skeleton and suggest that anabolic PTH may be a valuable therapeutic approach for autoimmune-associated skeletal disease.-Michalski, M. N., Seydel, A. L., Siismets, E. M., Zweifler, L. E., Koh, A. J., Sinder, B. P., Aguirre, J. I., Atabai, K., Roca, H., McCauley, L. K. Inflammatory bone loss associated with MFG-E8 deficiency is rescued by teriparatide.

Modulation of Osteoblastic Cell Efferocytosis by Bone Marrow Macrophages.

Apoptosis occurs at an extraordinary rate in the human body and the effective clearance of dead cells (efferocytosis) is necessary to maintain homeostasis and promote healing, yet the contribution and impact of this process in bone is unclear. Bone formation requires that bone marrow stromal cells (BMSCs) differentiate into osteoblasts which direct matrix formation and either become osteocytes, bone lining cells, or undergo apoptosis. A series of experiments were performed to identify the regulators and consequences of macrophage efferocytosis of apoptotic BMSCs (apBMSCs). Bone marrow derived macrophages treated with the anti-inflammatory cytokine interleukin-10 (IL-10) exhibited increased efferocytosis of apBMSCs compared to vehicle treated macrophages. Additionally, IL-10 increased anti-inflammatory M2-like macrophages (CD206+ ), and further enhanced efferocytosis within the CD206+ population. Stattic, an inhibitor of STAT3 phosphorylation, reduced the IL-10-mediated shift in M2 macrophage polarization and diminished IL-10-directed efferocytosis of apBMSCs by macrophages implicating the STAT3 signaling pathway. Cell culture supernatants and RNA from macrophages co-cultured with apoptotic bone cells showed increased secretion of monocyte chemotactic protein 1/chemokine (C-C motif) ligand 2 (MCP-1/CCL2) and transforming growth factor beta 1 (TGF-ß1) and increased ccl2 gene expression. In conclusion, IL-10 increases M2 macrophage polarization and enhances macrophage-mediated engulfment of apBMSCs in a STAT3 phosphorylation-dependent manner. After engulfment of apoptotic bone cells, macrophages secrete TGF-ß1 and MCP-1/CCL2, factors which fuel the remodeling process. A better understanding of the role of macrophage efferocytosis as it relates to normal and abnormal bone turnover will provide vital information for future therapeutic approaches to treat bone related diseases. J. Cell. Biochem. 117: 2697-2706, 2016. © 2016 Wiley Periodicals, Inc.

Polarization of prostate cancer-associated macrophages is induced by milk fat globule-EGF factor 8 (MFG-E8)-mediated efferocytosis.

Tumor cells secrete factors that modulate macrophage activation and polarization into M2 type tumor-associated macrophages, which promote tumor growth, progression, and metastasis. The mechanisms that mediate this polarization are not clear. Macrophages are phagocytic cells that participate in the clearance of apoptotic cells, a process known as efferocytosis. Milk fat globule- EGF factor 8 (MFG-E8) is a bridge protein that facilitates efferocytosis and is associated with suppression of proinflammatory responses. This study investigated the hypothesis that MFG-E8-mediated efferocytosis promotes M2 polarization. Tissue and serum exosomes from prostate cancer patients presented higher levels of MFG-E8 compared with controls, a novel finding in human prostate cancer. Coculture of macrophages with apoptotic cancer cells increased efferocytosis, elevated MFG-E8 protein expression levels, and induced macrophage polarization into an alternatively activated M2 phenotype. Administration of antibody against MFG-E8 significantly attenuated the increase in M2 polarization. Inhibition of STAT3 phosphorylation using the inhibitor Stattic decreased efferocytosis and M2 macrophage polarization in vitro, with a correlating increase in SOCS3 protein expression. Moreover, MFG-E8 knockdown tumor cells cultured with wild-type or MFG-E8-deficient macrophages resulted in increased SOCS3 expression with decreased STAT3 activation. This suggests that SOCS3 and phospho-STAT3 act in an inversely dependent manner when stimulated by MFG-E8 and efferocytosis. These results uncover a unique role of efferocytosis via MFG-E8 as a mechanism for macrophage polarization into tumor-promoting M2 cells.

Parathyroid hormone and trabectedin have differing effects on macrophages and stress fracture repair.

Stress fractures occur as a result of repeated mechanical stress on bone and are commonly found in the load-bearing lower extremities. Macrophages are key players in the immune system and play an important role in bone remodeling and fracture healing. However, the role of macrophages in stress fractures has not been adequately addressed. We hypothesize that macrophage infiltration into a stress fracture callus site promotes bone healing. To test this, a unilateral stress fracture induction model was employed in which the murine ulna of four-month-old, C57BL/6 J male mice was repeatedly loaded with a pre-determined force until the bone was displaced a distance below the threshold for complete fracture. Mice were treated daily with parathyroid hormone (PTH, 50 µg/kg/day) starting two days before injury and continued until 24 h before euthanasia either four or six days after injury, or treated with trabectedin (0.15 mg/kg) on the day of stress fracture and euthanized three or seven days after injury. These treatments were used due to their established effects on macrophages. While macrophages have been implicated in the anabolic effects of PTH, trabectedin, an FDA approved chemotherapeutic, compromises macrophage function and reduces bone mass. At three- and four-days post injury, callus macrophage numbers were analyzed histologically. There was a significant increase in macrophages with PTH treatment compared to vehicle in the callus site. By one week of healing, treatments differentially affected the bony callus as analyzed by microcomputed tomography. PTH enhanced callus bone volume. Conversely, callus bone volume was decreased with trabectedin treatment. Interestingly, concurrent treatment with PTH and trabectedin rescued the reduction observed in the callus with trabectedin treatment alone. This study reports on the key involvement of macrophages during stress fracture healing. Given these observed outcomes on macrophage physiology and bone healing, these findings may be important for patients actively receiving either of these FDA-approved therapeutics.

Patient-Reported Outcome Measurement and Minimal Clinically Important Difference for Hand Surgeons.

Patient-reported outcome measurement (PROM) tools are used to evaluate health status and response to treatment and have been integral in the effort to improve the quality of care provided. Patient reported outcomes (PROs) have garnered additional attention since becoming a priority of the National Institutes of Health in the early part of this century, and their use in both clinical practice and research has subsequently increased. In the upper extremity, a variety of PRO instruments exist that can assist physicians in their ability to track and/or prognosticate outcomes, make comparisons between treatments as well as strengthen research methodologies, and help determine the value of care. A more complete interpretation of the clinical significance of patient-reported outcome measurements is informed by parameters such as minimal clinically important difference, substantial clinical benefit and patient acceptable symptom state.

Caspase-9 driven murine model of selective cell apoptosis and efferocytosis.

Apoptosis and efficient efferocytosis are integral to growth, development, and homeostasis. The heterogeneity of these mechanisms in different cells across distinct tissues renders it difficult to develop broadly applicable in vivo technologies. Here, we introduced a novel inducible caspase-9 (iCasp9) mouse model which allowed targeted cell apoptosis and further facilitated investigation of concomitant efferocytosis. We generated iCasp9+/+ mice with conditional expression of chemically inducible caspase-9 protein that is triggered in the presence of Cre recombinase. In vitro, bone marrow cells from iCasp9+/+ mice showed expression of the iCasp9 protein when transduced with Cre-expressing adenovirus. Treatment of these cells with the chemical dimerizer (AP20187/AP) resulted in iCasp9 processing and cleaved caspase-3 upregulation, indicating successful apoptosis induction. The in vivo functionality and versatility of this model was demonstrated by crossing iCasp9+/+ mice with CD19-Cre and Osteocalcin (OCN)-Cre mice to target CD19+ B cells or OCN+ bone-lining osteoblasts. Immunofluorescence and/or immunohistochemical staining in combination with histomorphometric analysis of EGFP, CD19/OCN, and cleaved caspase-3 expression demonstrated that a single dose of AP effectively induced apoptosis in CD19+ B cells or OCN+ osteoblasts. Examination of the known efferocytes in the target tissues showed that CD19+ cell apoptosis was associated with infiltration of dendritic cells into splenic B cell follicles. In the bone, where efferocytosis remains under-explored, the use of iCasp9 provided direct in vivo evidence that macrophages are important mediators of apoptotic osteoblast clearance. Collectively, this study presented the first mouse model of iCasp9 which achieved selective apoptosis, allowing examination of subsequent efferocytosis. Given its unique feature of being controlled by any Cre-expressing mouse lines, the potential applications of this model are extensive and will bring forth more insights into the diversity of mechanisms and cellular effects induced by apoptosis including the physiologically important efferocytic process that follows.

Antisclerostin Effect on Osseointegration and Bone Remodeling.

Objective: This study reviewed the literature on local or systemic administration of antisclerostin, presenting results associated with osseointegration of dental/orthopedic implants and stimulation of bone remodeling. Materials and Methods: An extensive electronic search was conducted through MED-LINE/PubMed, PubMed Central, Web of Science databases and specific peer-reviewed journals to identify case reports, case series, randomized controlled trials, clinical trials and animal studies comparing either the systemic or local administration of antisclerostin and its effect in osseointegration and bone remodeling. Articles in English and with no restriction on period were included. Results: Twenty articles were selected for a full-text, and one was excluded. Finally, 19 articles were included in the study (16 animal studies and 3 randomized control trials). These studies were divided into two groups, which evaluated (i) osseointegration and (ii) bone remodeling potential. Initially 4560 humans and 1191 animals were identified. At least 1017 were excluded from the studies (981 humans and 36 animals), totaling 4724 subjects who completed (3579 humans and 1145 animals). (a) Osseointegration: 7 studies described this phenomenon; 4 reported bone-implant contact, which increased in all included studies. Similar results were found for bone mineral density, bone area/volume and bone thickness. (b) Bone remodeling: 13 studies were used for description. The studies reported an increase in BMD with sclerostin antibody treatment. A similar effect was found for bone mineral density/area/volume, trabecular bone and bone formation. Three biomarkers of bone formation were identified: bone-specific alkaline phosphatase (BSAP), osteocalcin and procollagen type 1 N-terminal Pro-peptide (P1NP); and markers for bone resorption were: serum C-telopeptide (sCTX), C-terminal telopeptides of type I collagen (CTX-1), ß-isomer of C-terminal telopeptides of type I collagen (ß-CTX) and tartrate-resistant acid phosphatase 5b (TRACP-5b). There were limitations: low number of human studies identified; high divergence in the model used (animal or human); the variance in the type of Scl-Ab and doses of administration; and the lack of reference quantitative values in the parameters analyzed by authors' studies (many articles only reported qualitative information). Conclusion: Within the limitations of this review and carefully observing all data, due to the number of articles included and the heterogeneity existing, more studies must be carried out to better evaluate the action of the antisclerostin on the osseointegration of dental implants. Otherwise, these findings can accelerate and stimulate bone remodeling and neoformation.

Chemical transfection of dye-conjugated microRNA precursors for microRNA functional analysis of M2 macrophages.

MicroRNAs (miRNAs) are short noncoding ribonucleic acids known to affect gene expression at the translational level and there is mounting evidence that miRNAs play a role in the function of tumor-associated macrophages (TAMs). To aid the functional analyses of miRNAs in an in-vitro model of TAMs known as M2 macrophages, a transfection method to introduce artificial miRNA constructs or miRNA molecules into primary human monocytes is needed. Unlike differentiated macrophages or dendritic cells, undifferentiated primary human monocytes have been known to show resistance to lentiviral transduction. To circumvent this challenge, other techniques such as electroporation and chemical transfection have been used in other applications to deliver small gene constructs into human monocytes. To date, no studies have compared these two methods objectively to evaluate their suitability in the miRNA functional analysis of M2 macrophages. Of the methods tested, the electroporation of miRNA-construct containing plasmids and the chemical transfection of miRNA precursor molecules are the most efficient approaches. The use of a silencer siRNA labeling kit (Ambion) to conjugate Cy 3 fluorescence dyes to the precursor molecules allowed the isolation of successfully transfected cells with fluorescence-activated cell sorting. The chemical transfection of these dye-conjugated miRNA precursors yield an efficiency of 37.5 ± 0.6% and a cell viability of 74 ± 1%. RNA purified from the isolated cells demonstrated good quality, and was fit for subsequent mRNA expression qPCR analysis. While electroporation of plasmids containing miRNA constructs yield transfection efficiencies comparable to chemical transfection of miRNA precursors, these electroporated primary monocytes seemed to have lost their potential for differentiation. Among the most common methods of transfection, the chemical transfection of dye-conjugated miRNA precursors was determined to be the best-suited approach for the functional analysis of M2 macrophages.

IL-4 induces proliferation in prostate cancer PC3 cells under nutrient-depletion stress through the activation of the JNK-pathway and survivin up-regulation.

Interleukin (IL)-4 plays a critical role in the regulation of immune responses and has been detected at high levels in the tumor microenvironment of cancer patients where it correlates with the grade of malignancy. The direct effect of IL-4 on cancer cells has been associated with increased cell survival; however, its role in cancer cell proliferation and related mechanisms is still unclear. Here it was shown that in a nutrient-depleted environment, IL-4 induces proliferation in prostate cancer PC3 cells. In these cells, under nutrient-depletion stress, IL-4 activates mitogen-activated protein kinases (MAPKs), including Erk, p38, and JNK. Using MAP-signaling-specific inhibitors, it was shown that IL-4-induced proliferation is mediated by JNK activation. In fact, JNK-inhibitor-V (JNKi-V) stunted IL-4-mediated cell proliferation. Furthermore, it was found that IL-4 induces survivin up-regulation in nutrient-depleted cancer cells. Using survivin-short-hairpin-RNAs (shRNAs), it was demonstrated that in this milieu survivin expression above a threshold limit is critical to the mechanism of IL-4-mediated proliferation. In addition, the significance of survivin up-regulation in a stressed environment was assessed in prostate cancer mouse xenografts. It was found that survivin knockdown decreases tumor progression in correlation with cancer cell proliferation. Furthermore, under nutrient depletion stress, IL -4 could induce proliferation in cancer cells from multiple origins: MDA-MB-231 (breast), A253 (head and neck), and SKOV-3 (ovarian). Overall, these findings suggest that in a tumor microenvironment under stress conditions, IL-4 triggers a simultaneous activation of the JNK-pathway and the up-regulation of survivin turning on a cancer proliferation mechanism.

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.

Cell-morphodynamic phenotype classification with application to cancer metastasis using cell magnetorotation and machine-learning.

We define cell morphodynamics as the cell's time dependent morphology. It could be called the cell's shape shifting ability. To measure it we use a biomarker free, dynamic histology method, which is based on multiplexed Cell Magneto-Rotation and Machine Learning. We note that standard studies looking at cells immobilized on microscope slides cannot reveal their shape shifting, no more than pinned butterfly collections can reveal their flight patterns. Using cell magnetorotation, with the aid of cell embedded magnetic nanoparticles, our method allows each cell to move freely in 3 dimensions, with a rapid following of cell deformations in all 3-dimensions, so as to identify and classify a cell by its dynamic morphology. Using object recognition and machine learning algorithms, we continuously measure the real-time shape dynamics of each cell, where from we successfully resolve the inherent broad heterogeneity of the morphological phenotypes found in a given cancer cell population. In three illustrative experiments we have achieved clustering, differentiation, and identification of cells from (A) two distinct cell lines, (B) cells having gone through the epithelial-to-mesenchymal transition, and (C) cells differing only by their motility. This microfluidic method may enable a fast screening and identification of invasive cells, e.g., metastatic cancer cells, even in the absence of biomarkers, thus providing a rapid diagnostics and assessment protocol for effective personalized cancer therapy.

CCL2 and interleukin-6 promote survival of human CD11b+ peripheral blood mononuclear cells and induce M2-type macrophage polarization.

CCL2 and interleukin (IL)-6 are among the most prevalent cytokines in the tumor microenvironment, with expression generally correlating with tumor progression and metastasis. CCL2 and IL-6 induced expression of each other in CD11b(+) cells isolated from human peripheral blood. It was demonstrated that both cytokines induce up-regulation of the antiapoptotic proteins cFLIP(L) (cellular caspase-8 (FLICE)-like inhibitory protein), Bcl-2, and Bcl-X(L) and inhibit the cleavage of caspase-8 and subsequent activation of the caspase-cascade, thus protecting cells from apoptosis under serum deprivation stress. Furthermore, both cytokines induced hyperactivation of autophagy in these cells. Upon CCL2 or IL-6 stimulation, CD11b(+) cells demonstrated a significant increase in the mannose receptor (CD206) and the CD14(+)/CD206(+) double-positive cells, suggesting a polarization of macrophages toward the CD206(+) M2-type phenotype. Caspase-8 inhibitors mimicked the cytokine-induced up-regulation of autophagy and M2 polarization. Furthermore, E64D and leupeptin, which are able to function as inhibitors of autophagic degradation, reversed the effect of caspase-8 inhibitors in the M2-macrophage polarization, indicating a role of autophagy in this mechanism. Additionally, in patients with advanced castrate-resistant prostate cancer, metastatic lesions exhibited an increased CD14(+)/CD206(+) double-positive cell population compared with normal tissues. Altogether, these findings suggest a role for CCL2 and IL-6 in the survival of myeloid monocytes recruited to the tumor microenvironment and their differentiation toward tumor-promoting M2-type macrophages via inhibition of caspase-8 cleavage and enhanced autophagy.

Identification and functional characterization of ERK/MAPK phosphorylation sites in the Runx2 transcription factor.

The Runx2 transcription factor is required for commitment of mesenchymal cells to bone lineages and is a major regulator of osteoblast-specific gene expression. Runx2 is subject to a number of post-transcriptional controls including selective proteolysis and phosphorylation. We previously reported that Runx2 is phosphorylated and activated by the ERK/MAPK pathway (Xiao, G., Jiang, D., Thomas, P., Benson, M. D., Guan, K., Karsenty, G., and Franceschi, R. T. (2000) J. Biol. Chem. 275, 4453-4459). In this study, we used a combination of in vitro and in vivo phosphorylation analysis, mass spectroscopy, and functional assays to identify two sites at Ser(301) and Ser(319) within the proline/serine/threonine domain of Runx2 that are required for this regulation. These sites are phosphorylated by activated ERK1 in vitro and in cell culture. In addition to confirming ERK-dependent phosphorylation at Ser(319), mass spectroscopy identified two other ERK-phosphorylated sites at Ser(43) and Ser(510). Furthermore, introduction of S301A,S319A mutations rendered Runx2 resistant to MAPK-dependent activation and reduced its ability to stimulate osteoblast-specific gene expression and differentiation after transfection into Runx2-null calvarial cells and mesenchymal cells. In contrast, S301E,S319E Runx2 mutants had enhanced transcriptional activity that was minimally dependent on MAPK signaling, consistent with the addition of a negative charge mimicking serine phosphorylation. These results emphasize the important role played by Runx2 phosphorylation in the control of osteoblast gene expression and provide a mechanism to explain how physiological signals acting on bone through the ERK/MAPK pathway can stimulate osteoblast-specific gene expression.

Analysis of transcription factor interactions in osteoblasts using competitive chromatin immunoprecipitation.

Chromatin immunoprecipitation (ChIP) is a widely used technique for quantifying protein-DNA interactions in living cells. This method commonly uses fixed (crosslinked) chromatin that is fragmented by sonication (X-ChIP). We developed a simple new ChIP procedure for the immunoprecipitation of sonicated chromatin isolated from osteoblasts in the absence of crosslinking (N-ChIP). The use of noncrosslinked chromatin allowed development of a new modification of the ChIP assay: the combination of N-ChIP and competition with double-stranded oligonucleotides containing specific binding sites for individual transcription factors (Competitive N-ChIP). Using this approach, we were able to discriminate between individual binding sites for the Runx2 transcription factor in the osteocalcin and bone sialoprotein genes that cannot be resolved by traditional X-ChIP. N-ChIP assays were also able to detect several other types of chromatin interactions including those with Dlx homeodomain factors and nuclear proteins such as Sin3a that lack an intrinsic DNA-binding motif and, therefore, bind to chromatin via interactions with other proteins.

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.

Transcriptional regulation of osteoblasts.

The differentiation of osteoblasts from mesenchymal precursors requires a series of cell fate decisions controlled by a hierarchy of transcription factors. These include RUNX2, Osterix (OSX), ATF4 and a large number of nuclear coregulators. During bone development, initial RUNX2 expression coincides with the formation of mesenchymal condensations and precedes the branching of chondrogenic and osteogenic lineages. Given its central role in bone development, it is not surprising that RUNX2 is subject to a variety of controls. These include posttranslational modification, especially phosphorylation, and interactions with accessory nuclear factors. Specific examples of RUNX2 regulation to be reviewed include phosphorylation by the ERK/MAP kinase pathway and interactions with DLX5. RUNX2 is regulated via phosphorylation of critical serine residues in the proline/serine/threonine domain. In vivo, the transgenic expression of constitutively active MAP kinase in osteoblasts accelerated skeletal development, while a dominant-negative MAPK retarded development in a RUNX2-dependent manner. DLX5-RUNX2 complexes can be detected in osteoblasts and this interaction plays a critical role in maintaining osteoblast-specific expression of the bone sialoprotein gene. These studies allow us to begin understanding the complex mechanisms necessary to fine-tune bone formation as mesenchymal progenitors progress down the osteoblast lineage.