van Wijnen, Andre J

Serum amyloid A (A‐SAA/Saa3) was shown before to affect osteoblastic metabolism. Here, using RT‐quantitative PCR and/or immunoblotting, we show that expression of mouse Saa3 and human SAA1 and SAA2 positively correlates with increased cellular maturation toward the osteocyte phenotype. Expression is not detected in C3H10T1/2 embryonic fibroblasts but is successively higher in preosteoblastic MC3T3‐E1 cells, late osteoblastic MLO‐A5 cells, and MLO‐Y4 osteocytes, consistent with findings using primary bone cells from newborn mouse calvaria. Recombinant Saa3 protein functionally inhibits osteoblast differentiation as reflected by reductions in the expression of osteoblast markers and decreased mineralization in newborn mouse calvaria. Yet, Saa3 protein enhances osteoclastogenesis in mouse macrophages/monocytes based on the number of multinucleated and tartrate‐resistant alkaline phosphatase‐positive cells and Calcr mRNA expression. Depletion of Saa3 in MLO osteocytes results in the loss of the mature osteocyte phenotype. Recombinant osteocalcin, which is reciprocally regulated with Saa3 at the osteoblast/osteocyte transition, attenuates Saa3 expression in MLO‐Y4 osteocytes. Mechanistically, Saa3 produced by MLO‐Y4 osteocytes is integrated into the extracellular matrix of MC3T3‐E1 osteoblasts, where it associates with the P2 purinergic receptor P2rx7 to stimulate Mmp13 expression via the P2rx7/MAPK/ERK/activator protein 1 axis. Our data suggest that Saa3 may function as an important coupling factor in bone development and homeostasis.—Thaler, R., Sturmlechner, I., Spitzer, S., Riester, S. M., Rumpler, M., Zwerina, J., Klaushofer, K., vanWijnen, A. J., Varga, F. Acute‐phase protein serum amyloid A3 is a novel paracrine coupling factor that controls bone homeostasis. FASEB J. 29, 1344‐1359 (2015). www.fasebj.org

The biological interface between an orthopedic implant and the surrounding host tissue may have a dramatic effect upon clinical outcome. Desired effects include bony ingrowth (osseointegration), stimulation of osteogenesis (osteoinduction), increased vascularization, and improved mechanical stability. Implant loosening, fibrous encapsulation, corrosion, infection, and inflammation, as well as physical mismatch may have deleterious clinical effects. This is particularly true of implants used in the reconstruction of load-bearing synovial joints such as the knee, hip, and the shoulder. The surfaces of orthopedic implants have evolved from solid-smooth to roughened-coarse and most recently, to porous in an effort to create a three-dimensional architecture for bone apposition and osseointegration. Total joint surgeries are increasingly performed in younger individuals with a longer life expectancy, and therefore, the postimplantation lifespan of devices must increase commensurately. This review discusses advancements in biomaterials science and cell-based therapies that may further improve orthopedic success rates. We focus on material and biological properties of orthopedic implants fabricated from porous metal and highlight some relevant developments in stem-cell research. We posit that the ideal primary and revision orthopedic load-bearing metal implants are highly porous and may be chemically modified to induce stem cell growth and osteogenic differentiation, while minimizing inflammation and infection. We conclude that integration of new biological, chemical, and mechanical methods is likely to yield more effective strategies to control and modify the implant–bone interface and thereby improve long-term clinical outcomes.

ABSTRACT Exposure of bone to dynamic strain increases the rate of division of osteoblasts and also influences the directional organization of the cellular and molecular structure of the bone tissue that they produce. Here, we report that brief exposure to dynamic substrate strain (sufficient to rapidly stimulate cell division) influences the orientation of osteoblastic cell division. The initial proliferative response to strain involves canonical Wnt signaling and can be blocked by sclerostin. However, the strain-related orientation of cell division is independently influenced through the noncanonical Wnt/planar cell polarity (PCP) pathway. Blockade of Rho-associated coiled kinase (ROCK), a component of the PCP pathway, prevents strain-related orientation of division in osteoblast-like Saos-2 cells. Heterozygous loop-tail mutation of the core PCP component van Gogh-like 2 (Vangl2) in mouse osteoblasts impairs the orientation of division in response to strain. Examination of bones from Vangl2 loop-tail heterozygous mice by µCT and scanning electron microscopy reveals altered bone architecture and disorganized bone-forming surfaces. Hence, in addition to the well-accepted role of PCP involvement in response to developmental cues during skeletal morphogenesis, our data reveal that this pathway also acts postnatally, in parallel with canonical Wnt signaling, to transduce biomechanical cues into skeletal adaptive responses. The simultaneous and independent actions of these two pathways appear to influence both the rate and orientation of osteoblast division, thus fine-tuning bone architecture to meet the structural demands of functional loading. © 2014 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals, Inc. on behalf of the American Society for Bone and Mineral Research.

ABSTRACTMitotic bookmarking is an epigenetic control mechanism that sustains gene expression in progeny cells; it is often found in genes related to the maintenance of cellular phenotype and growth control. RUNX transcription factors regulate a broad spectrum of RNA Polymerase (Pol II) transcribed genes important for lineage commitment but also regulate RNA Polymerase I (Pol I) driven ribosomal gene expression, thus coordinating control of cellular identity and proliferation. In this study, using fluorescence microscopy and biochemical approaches we show that the principal RUNX co‐factor, CBFβ, associates with nucleolar organizing regions (NORs) during mitosis to negatively regulate RUNX‐dependent ribosomal gene expression. Of clinical relevance, we establish for the first time that the leukemogenic fusion protein CBFβ‐SMMHC (smooth muscle myosin heavy chain) also associates with ribosomal genes in interphase chromatin and mitotic chromosomes to promote and epigenetically sustain regulation of ribosomal genes through RUNX factor interactions. Our results demonstrate that CBFβ contributes to the transcriptional regulation of ribosomal gene expression and provide further understanding of the epigenetic role of CBFβ‐SMMHC in proliferation and maintenance of the leukemic phenotype. J. Cell. Biochem. 115: 2155–2164, 2014. © 2014 Wiley Periodicals, Inc.

Human adipose‐derived mesenchymal stromal cells (AMSCs) grown in platelet lysate are promising agents for therapeutic tissue regeneration. Here, we investigated whether manipulation of epigenetic events by the clinically relevant histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA) alters differentiation of AMSCs. The multipotency of AMSCs was validated by their ability to differentiate into osteogenic, chondrogenic, and adipogenic lineages. High‐throughput RNA sequencing and RT‐qPCR established that human histone deacetylases (HDAC1 to HDAC11, and SIRT1 to SIRT7) are differentially expressed in AMSCs. SAHA induces hyper‐acetylation of histone H3 and H4, stimulates protein expression of the HDAC‐responsive gene SLC9A3R1/NHERF1 and modulates the AKT/FOXO1 pathway. Biologically, SAHA interferes with osteogenic, chondrogenic and adipogenic lineage commitment of multipotent AMSCs. Mechanistically, SAHA‐induced loss of differentiation potential of uncommitted AMSCs correlates with multiple changes in the expression of principal transcription factors that control mesenchymal or pluripotent states. We propose that SAHA destabilizes the multi‐potent epigenetic state of uncommitted human AMSCs by hyper‐acetylation and perturbation of key transcription factor pathways. Furthermore, AMSCs grown in platelet lysate may provide a useful biological model for screening of new HDAC inhibitors that control the biological fate of human mesenchymal stromal cells. J. Cell. Physiol. 230: 52–62, 2015. © 2014 Wiley Periodicals, Inc.

AbstractImproving the effectiveness of adipose‐tissue derived human mesenchymal stromal/stem cells (AMSCs) for skeletal therapies requires a detailed characterization of mechanisms supporting cell proliferation and multi‐potency. We investigated the molecular phenotype of AMSCs that were either actively proliferating in platelet lysate or in a basal non‐proliferative state. Flow cytometry combined with high‐throughput RNA sequencing (RNASeq) and RT‐qPCR analyses validate that AMSCs express classic mesenchymal cell surface markers (e.g., CD44, CD73/NT5E, CD90/THY1, and CD105/ENG). Expression of CD90 is selectively elevated at confluence. Self‐renewing AMSCs express a standard cell cycle program that successively mediates DNA replication, chromatin packaging, cyto‐architectural enlargement, and mitotic division. Confluent AMSCs preferentially express genes involved in extracellular matrix (ECM) formation and cellular communication. For example, cell cycle‐related biomarkers (e.g., cyclins E2 and B2, transcription factor E2F1) and histone‐related genes (e.g., H4, HINFP, NPAT) are elevated in proliferating AMSCs, while ECM genes are strongly upregulated (>10‐fold) in quiescent AMSCs. AMSCs also express pluripotency genes (e.g., POU5F1, NANOG, KLF4) and early mesenchymal markers (e.g., NES, ACTA2) consistent with their multipotent phenotype. Strikingly, AMSCs modulate expression of WNT signaling components and switch production of WNT ligands (from WNT5A/WNT5B/WNT7B to WNT2/WNT2B), while upregulating WNT‐related genes (WISP2, SFRP2, and SFRP4). Furthermore, post‐proliferative AMSCs spontaneously express fibroblastic, osteogenic, chondrogenic, and adipogenic biomarkers when maintained in confluent cultures. Our findings validate the biological properties of self‐renewing and multi‐potent AMSCs by providing high‐resolution quality control data that support their clinical versatility. J. Cell. Biochem. 115: 1816–1828, 2014. © 2014 Wiley Periodicals, Inc.

Understanding the mechanisms that sustain pluripotency in human embryonic stem cells (hESCs) is an active area of research that may prove useful in regenerative medicine and will provide fundamental information relevant to development and cancer. hESCs and cancer cells share the unique ability to proliferate indefinitely and rapidly. Because the protein survivin is uniquely overexpressed in virtually all human cancers and in hESCs, we sought to investigate its role in supporting the distinctive capabilities of these cell types. Results presented here suggest that survivin contributes to the maintenance of pluripotency and that post‐transcriptional control of survivin isoform expression is selectively regulated by microRNAs. miR‐203 has been extensively studied in human tumors, but has not been characterized in hESCs. We show that miR‐203 expression and activity is consistent with the expression and subcellular localization of survivin isoforms that in turn modulate expression of the Oct4 and Nanog transcription factors to sustain pluripotency. This study contributes to understanding of the complex regulatory mechanisms that govern whether hESCs proliferate or commit to lineages. J. Cell. Physiol. 230: 63–70, 2015. © 2014 Wiley Periodicals, Inc.

Ewing sarcoma is an aggressive pediatric small round cell tumor that predominantly occurs in bone. Approximately 85% of Ewing sarcomas harbor the EWS/FLI fusion protein, which arises from a chromosomal translocation, t(11:22)(q24:q12). EWS/FLI interacts with numerous lineage‐essential transcription factors to maintain mesenchymal progenitors in an undifferentiated state. We previously showed that EWS/FLI binds the osteogenic transcription factor RUNX2 and prevents osteoblast differentiation. In this study, we investigated the role of another Runt‐domain protein, RUNX3, in Ewing sarcoma. RUNX3 participates in mesenchymal‐derived bone formation and is a context dependent tumor suppressor and oncogene. RUNX3 was detected in all Ewing sarcoma cells examined, whereas RUNX2 was detected in only 73% of specimens. Like RUNX2, RUNX3 binds to EWS/FLI via its Runt domain. EWS/FLI prevented RUNX3 from activating the transcription of a RUNX‐responsive reporter, p6OSE2. Stable suppression of RUNX3 expression in the Ewing sarcoma cell line A673 delayed colony growth in anchorage independent soft agar assays and reversed expression of EWS/FLI‐responsive genes. These results demonstrate an important role for RUNX3 in Ewing sarcoma. J. Cell. Physiol. 229: 2049–2056, 2014. © 2014 Wiley Periodicals, Inc.

ABSTRACTThe chromatin remodeling complex SWI/SNF and the transcription factor C/EBPβ play critical roles in osteoblastic cells as they jointly control transcription of a number of bone‐related target genes. The largest C/EBPβ isoform, LAP*, possesses a short additional N‐terminal domain that has been proposed to mediate the interaction of this factor with SWI/SNF in myeloid cells. Here we examine the requirement of a functional N‐terminus in C/EBPβ‐LAP* for binding SWI/SNF and for recruiting this complex to the Ric‐8B gene to mediate transcriptional repression. We find that both C/EBPβ‐LAP* and SWI/SNF simultaneously bind to the Ric‐8B promoter in differentiating osteoblasts that repress Ric‐8B expression. This decreased expression of Ric‐8B is not accompanied by significant changes in histone acetylation at the Ric‐8B gene promoter sequence. A single aminoacid change at the C/EBPβ‐LAP* N‐terminus (R3L) that inhibits C/EBPβ‐LAP*‐SWI/SNF interaction, also prevents SWI/SNF recruitment to the Ric‐8B promoter as well as C/EBPβ‐LAP*‐dependent repression of the Ric‐8B gene. Inducible expression of the C/EBPβ‐LAP*R3L protein in stably transfected osteoblastic cells demonstrates that this mutant protein binds to C/EBPβ‐LAP*‐target promoters and competes with the endogenous C/EBPβ factor. Together our results indicate that a functional N‐terminus in C/EBPβ‐LAP* is required for interacting with SWI/SNF and for Ric‐8B gene repression in osteoblasts. J. Cell. Physiol. 229: 1521–1528, 2014. © 2014 Wiley Periodicals, Inc.