Westendorf, Jennifer J.

The mechanical properties of biological tissues influence their function and can predict degenerative conditions before gross histological or physiological changes are detectable. This is especially true for structural tissues such as articular cartilage, which has a primarily mechanical function that declines after injury and in the early stages of osteoarthritis. While atomic force microscopy (AFM) has been used to test the elastic modulus of articular cartilage before, there is no agreement or consistency in methodologies reported. For murine articular cartilage, methods differ in two major ways: experimental parameter selection and sample preparation. Experimental parameters that affect AFM results include indentation force and cantilever stiffness; these are dependent on the tip, sample, and instrument used. The aim of this project was to optimize these experimental parameters to measure murine articular cartilage elastic modulus by AFM micro-indentation. We first investigated the effects of experimental parameters on a control material, polydimethylsiloxane gel (PDMS), which has an elastic modulus on the same order of magnitude as articular cartilage. Experimental parameters were narrowed on this control material, and then finalized on wildtype C57BL/6J murine articular cartilage samples that were prepared with a novel technique that allows for cryosectioning of epiphyseal segments of articular cartilage and long bones without decalcification. This technique facilitates precise localization of AFM measurements on the murine articular cartilage matrix and eliminates the need to separate cartilage from underlying bone tissues, which can be challenging in murine bones because of their small size. Together, the new sample preparation method and optimized experimental parameters provide a reliable standard operating procedure to measure microscale variations in the elastic modulus of murine articular cartilage.

Abstract Osteoblasts are known to protect acute myeloid leukemia (AML) cells in the bone marrow microenvironment, however, the identity of these protective signals is not known. Because disrupting this protection could help prevent the relapse of AML patients following chemotherapy, we characterized the molecular mechanisms. We previously showed that SDF-1 (CXCL12), a chemokine abundant in the bone marrow, induces apoptosis of AML cells in cocultures unless the leukemic cells are protected via proximity to differentiating osteoblasts. Treating differentiating osteoblasts with either suberoylanilide hydroxamic acid (SAHA) or LBH-589 histone deacetylase (HDAC) inhibitor drugs inhibited their protection of AML cells. RNAseq revealed that SAHA globally inhibits osteoblast differentiation while inducing expression of the scaffold protein, Nherf-1. Remarkably, expressing Nherf-1 in differentiating osteoblasts was both required and sufficient to block osteoblast-mediated protection of AML cells. Together, these results indicate that disrupting the differentiation of osteoprogenitors in the bone marrow inhibits their ability to protect AML cells from apoptosis, and that this can be achieved via either inhibiting HDACs or overexpressing Nherf-1 in osteoblast-lineage cells. HDAC inhibitors or treatments specifically designed to induce Nherf-1 expression either during or immediately prior to chemotherapy may therefore be a useful strategy for reducing the risk of disease relapse in AML patients.

Abstract Expression of autocrine growth factors by myeloma cells is an important mechanism that may contribute to tumor expansion. IL-6 is one of several cytokines that uses the signal transducer gp130 as a receptor component. Of these cytokines, those that have been shown to be paracrine growth factors for some myeloma cells include IL-6, IL-11, ciliary neurotrophic factor, leukemia inhibitory factor, and oncostatin M (OSM). Only IL-6, however, has been identified as an autocrine growth factor for myeloma cells. In this study we used a panel of three IL-6-responsive myeloma cell lines to investigate the expression of other autocrine growth factor loops. Initial studies employing neutralizing mAbs to IL-6 or gp130 revealed that the growth of the DP-6 and KP-6 cell lines was inhibited by both mAbs, whereas the growth of the KAS-6/1 cell line was inhibited only by the anti-gp130 mAb. Anti-OSM neutralizing mAb also inhibited KAS-6/1 cell growth. Autocrine OSM production by the KAS-6/1 cells was confirmed using a sensitive ELISA. Although the anti-OSM mAb had no significant effects on KP-6 and DP-6 cell growth, OSM was detected in DP-6 supernatants. These results suggest that OSM production and responsiveness by myeloma cells are distinct phenotypes and not necessarily related in all myeloma cells. Finally, we analyzed the significance of OSM-mediated myeloma cell growth by assessing the effects of OSM on normal, in vitro-generated plasmablasts. OSM markedly enhanced plasmablast Ig secretion but did not affect growth. Thus, the nature of the response elicited by OSM in myeloma cells is distinct from its effects on normal B lineage cells. Moreover, because gp130-mediated signaling results in myeloma cell growth, autocrine expression of any gp130-utilizing cytokine has the potential to significantly augment tumor expansion.

Abstract Myeloma is a neoplasia characterized by the accumulation of malignant plasma cells in the bone marrow. In these studies, we have demonstrated that CD40 is expressed in human myeloma cells and have used a recently established IL-6-dependent myeloma cell line, ANBL-6, to examine the potential function of CD40 expression in myeloma cells. In addition to its expression on the ANBL-6 cells, we show that CD40 is expressed on freshly isolated myeloma cells from seven of seven patients tested. To address the role of CD40 expression in myeloma cells, we have examined the responsiveness of the ANBL-6 cell line to a CD40-specific mAb, G28-5. This cell line has previously been shown to proliferate only in response to IL-6. Of interest in this study, G28-5 also induced proliferation of the ANBL-6 cells. This proliferation was substantially inhibited by an IL-6-neutralizing mAb. Analysis of ANBL-6 cell culture supernatants by ELISA demonstrated that G28-5-stimulated cells secreted significant levels of IL-6, whereas unstimulated cell culture supernatants contained undetectable levels of IL-6. Furthermore, CV-1/EBNA cells expressing the human CD40 ligand also induced the proliferation of the ANBL-6 cell line, an effect that was inhibited by the anti-IL-6 mAb. Lastly, RNA blot analysis demonstrated an increase in IL-6 message in G28-5-stimulated ANBL-6 cells over unstimulated cells. These results indicate that the primary mechanism of anti-CD40-stimulated proliferation of the ANBL-6 cells is the induction of autocrine IL-6 production. Moreover, these data suggest that the expression of CD40 in malignant plasma cells may play a role in tumor cell expansion, possibly by stimulating the induction of autocrine IL-6 secretion.

Abstract The Journal of Bone and Mineral Research (JBMR®), the flagship journal of the American Society for Bone and Mineral Research (ASBMR), enjoys a premiere position in its field and has a global reach. The journal uses a single-blind peer-review process whereby three editors are typically involved in assessing each submission for publication, in addition to external reviewers. Although emphasizing fairness, rigor, and transparency, this process is not immune to the influence of unconscious biases. The gender and geographic diversity of JBMR® authors, editors, and reviewers has increased over the last three decades, but whether such diversity has affected peer-review outcomes is unknown. We analyzed manuscript acceptance rates based on the gender and geographic origin of authors, reviewers, and Associate Editors. The analysis included 1662 original research articles submitted to JBMR® from September 2017 through December 2019. Gender was assigned using probabilities from an online tool and manually validated through internet searches. Predictor variables of manuscript outcome were determined with multivariate logistic regression analysis. The acceptance rate was highest when the first and last authors were of different genders, and lowest when both authors were men. Reviewer gender did not influence the outcome regardless of the genders of the first and last authors. Associate Editors from all geographical regions tended to select reviewers from their same region. The acceptance rate was highest when the Associate Editor was from Europe. Manuscripts with authors from North America and Australia/New Zealand had greater overall odds of acceptance than those from Europe and Asia. Manuscripts reviewed only by Editorial Board (EB) members had a lower acceptance rate than those refereed by non-EB reviewers or a mix of EB and non-EB reviewers. Overall, the geographical origin of authors, reviewers, and editors, as well as reviewers' EB membership may influence manuscript decisions. Yet, the JBMR® peer-review process remains largely free from gender bias. © 2022 American Society for Bone and Mineral Research (ASBMR).

Abstract One of the greatest barriers to curative treatment of neuroblastoma is its frequent metastatic outgrowth prior to diagnosis, especially in cases driven by amplification of the MYCN oncogene. However, only a limited number of regulatory proteins that contribute to this complex MYCN-mediated process have been elucidated. Here we show that the growth arrest-specific 7 (GAS7) gene, located at chromosome band 17p13.1, is preferentially deleted in high-risk MYCN-driven neuroblastoma. GAS7 expression was also suppressed in MYCN-amplified neuroblastoma lacking 17p deletion. GAS7 deficiency led to accelerated metastasis in both zebrafish and mammalian models of neuroblastoma with overexpression or amplification of MYCN. Analysis of expression profiles and the ultrastructure of zebrafish neuroblastoma tumors with MYCN overexpression identified that GAS7 deficiency led to (i) downregulation of genes involved in cell–cell interaction, (ii) loss of contact among tumor cells as critical determinants of accelerated metastasis, and (iii) increased levels of MYCN protein. These results provide the first genetic evidence that GAS7 depletion is a critical early step in the cascade of events culminating in neuroblastoma metastasis in the context of MYCN overexpression. Significance: Heterozygous deletion or MYCN-mediated repression of GAS7 in neuroblastoma releases an important brake on tumor cell dispersion and migration to distant sites, providing a novel mechanism underlying tumor metastasis in MYCN-driven neuroblastoma. See related commentary by Menard, p. 2815

ABSTRACT Endochondral ossification is tightly controlled by a coordinated network of signaling cascades including parathyroid hormone (PTH). Pleckstrin homology (PH) domain and leucine rich repeat phosphatase 1 (Phlpp1) affects endochondral ossification by suppressing chondrocyte proliferation in the growth plate, longitudinal bone growth, and bone mineralization. As such, Phlpp1−/− mice have shorter long bones, thicker growth plates, and proportionally larger growth plate proliferative zones. The goal of this study was to determine how Phlpp1 deficiency affects PTH signaling during bone growth. Transcriptomic analysis revealed greater PTH receptor 1 (Pth1r) expression and enrichment of histone 3 lysine 27 acetylation (H3K27ac) at the Pth1r promoter in Phlpp1-deficient chondrocytes. PTH (1-34) enhanced and PTH (7-34) attenuated cell proliferation, cAMP signaling, cAMP response element-binding protein (CREB) phosphorylation, and cell metabolic activity in Phlpp1-inhibited chondrocytes. To understand the role of Pth1r action in the endochondral phenotypes of Phlpp1-deficient mice, Phlpp1−/− mice were injected with Pth1r ligand PTH (7-34) daily for the first 4 weeks of life. PTH (7-34) reversed the abnormal growth plate and long-bone growth phenotypes of Phlpp1−/− mice but did not rescue deficits in bone mineral density or trabecular number. These results show that elevated Pth1r expression and signaling contributes to increased proliferation in Phlpp1−/− chondrocytes and shorter bones in Phlpp1-deficient mice. Our data reveal a novel molecular relationship between Phlpp1 and Pth1r in chondrocytes during growth plate development and longitudinal bone growth. © 2021 American Society for Bone and Mineral Research (ASBMR).