Jason W. Ashley

Calcium-independent phospholipases A2 and their roles in biological processes and diseases

Among the family of phospholipases A2 (PLA2s) are the Ca2+-independent PLA2s (iPLA2s) and they are designated group VI iPLA2s. In relation to secretory and cytosolic PLA2s, the iPLA2s are more recently described and details of their expression and roles in biological functions are rapidly emerging. The iPLA2s or patatin-like phospholipases (PNPLAs) are intracellular enzymes that do not require Ca2+ for activity, and contain lipase (GXSXG) and nucleotide-binding (GXGXXG) consensus sequences. Though nine PNPLAs have been recognized, PNPLA8 (membrane-associated iPLA2γ) and PNPLA9 (cytosol-associated iPLA2β) are the most widely studied and understood. The iPLA2s manifest a variety of activities in addition to phospholipase, are ubiquitously expressed, and participate in a multitude of biological processes, including fat catabolism, cell differentiation, maintenance of mitochondrial integrity, phospholipid remodeling, cell proliferation, signal transduction, and cell death. As might be expected, increased or decreased expression of iPLA2s can have profound effects on the metabolic state, CNS function, cardiovascular performance, and cell survival; therefore, dysregulation of iPLA2s can be a critical factor in the development of many diseases. This review is aimed at providing a general framework of the current understanding of the iPLA2s and discussion of the potential mechanisms of action of the iPLA2s and related involved lipid mediators.

Selective targeting of RANK signaling pathways as new therapeutic strategies for osteoporosis

Importance of the field: Osteoporosis has become a worldwide health and social issue due to an aging population. Four major antiresorptive drugs (agents capable of inhibiting osteoclast formation and/or function) are currently available on the market: estrogen, selective estrogen receptor modulators (SERMs), bisphosphonates and calcitonin. These drugs either lack satisfactory efficacy or have potential to cause serious side effects. Thus, development of more efficacious and safer drugs is warranted. Areas covered in this review: The discovery of the receptor activator of NF-κB ligand (RANKL) and its two receptors, RANK and osteoprotegerin (OPG), has not only established a crucial role for the RANKL/RANK/OPG axis in osteoclast biology but also created a great opportunity to develop new drugs targeting this system for osteoporosis therapy. This review focuses on discussion of therapeutic targeting of RANK signaling. What the reader will gain: An update on the functions of RANKL and an overview of the known RANK signaling pathways in osteoclasts. A discussion of rationales for exploring RANK signaling pathways as potent and specific therapeutic targets to promote future development of better drugs for osteoporosis. Take home message: Several RANK signaling components have the potential to serve as potent and specific therapeutic targets for osteoporosis.

Molecular basis of requirement of receptor activator of nuclear factor κB signaling for interleukin 1-mediated osteoclastogenesis.

Background: Interleukin 1-mediated osteoclastogenesis requires permissive levels of RANKL or RANKL pretreatment. Results: Interleukin 1 can only activate the expression of osteoclast markers and osteoclastogenic transcription factor NFATc1 with permissive levels of RANKL or RANKL pretreatment. Conclusion: RANKL is involved in interleukin 1-mediated osteoclastogenesis by rendering osteoclast and NFATc1 genes responsive to interleukin 1. Significance: This is a novel mechanism of interleukin 1-mediated osteoclastogenesis. IL-1, a proinflammatory cytokine, is implicated in bone loss in various pathological conditions by promoting osteoclast formation, survival, and function. Although IL-1 alone can sufficiently prolong osteoclast survival and activate osteoclast function, IL-1-mediated osteoclastogenesis requires the receptor activator of NF-κB (RANK) ligand (RANKL). However, the molecular basis of the dependence of IL-1-mediated osteoclastogenesis on RANKL is not fully understood. Here we show that although IL-1 cannot activate the expression of the osteoclast genes encoding matrix metalloproteinase 9, cathepsin K, tartrate-resistant acid phosphatase, and carbonic anhydrase II in bone marrow macrophages (BMMs), RANKL renders these osteoclast genes responsive to IL-1. We further demonstrate that IL-1 alone fails to induce the expression of nuclear factor of activated T cell cytoplasmic 1 (NFATc1), a master transcriptional regulator of osteoclastogenesis), in BMMs but can up-regulate its expression in the presence of permissive levels of RANKL or with RANKL pretreatment. The RANK IVVY motif, which has been previously shown to commit BMMs to the osteoclast lineage in RANKL- and TNF α-mediated osteoclastogenesis, also plays a crucial role in IL-1-mediated osteoclastogenesis by changing the four osteoclast marker and NFATc1 genes to an IL-1-inducible state. Finally, we show that MyD88, a known critical component of the IL-1 receptor I signaling pathway, plays a crucial role in IL-1-mediated osteoclastogenesis from RANKL-primed BMMs by up-regulating the expression of the osteoclast marker and NFATc1 genes. This study reveals a novel mechanism of IL-1-mediated osteoclastogenesis and supports the promising potential of the IVVY motif to serve as a therapeutic target for inflammatory bone loss.

c-Src Links a RANK/αvβ3 Integrin Complex to the Osteoclast Cytoskeleton

ABSTRACT RANK ligand (RANKL), by mechanisms unknown, directly activates osteoclasts to resorb bone. Because c-Src is key to organizing the cells cytoskeleton, we asked if the tyrosine kinase also mediates RANKL-stimulated osteoclast activity. RANKL induces c-Src to associate with RANK369–373 in an αvβ3-dependent manner. Furthermore, RANK369–373 is the only one of six putative TRAF binding motifs sufficient to generate actin rings and activate the same cytoskeleton-organizing proteins as the integrin. While c-Src organizes the cells cytoskeleton in response to the cytokine, it does not participate in RANKL-stimulated osteoclast formation. Attesting to their collaboration, αvβ3 and activated RANK coprecipitate, but only in the presence of c-Src. c-Src binds activated RANK via its Src homology 2 (SH2) domain and αvβ3 via its SH3 domain, suggesting the kinase links the two receptors. Supporting this hypothesis, deletion or inactivating point mutation of either the c-Src SH2 or SH3 domain obviates the RANK/αvβ3 association. Thus, activated RANK prompts two distinct signaling pathways; one promotes osteoclast formation, and the other, in collaboration with c-Src-mediated linkage to αvβ3, organizes the cells cytoskeleton.

Genetic Ablation of CD68 Results in Mice with Increased Bone and Dysfunctional Osteoclasts

CD68 is a member of the lysosome associated membrane protein (LAMP) family that is restricted in its expression to cells of the monocyte/macrophage lineage. This lineage restriction includes osteoclasts, and, while previous studies of CD68 in macrophages and dendritic cells have proposed roles in lipid metabolism, phagocytosis, and antigen presentation, the expression and function of CD68 in osteoclasts have not been explored. In this study, we investigated the expression and localization of CD68 in macrophages and osteoclasts in response to the monocyte/macrophage-colony stimulating factor (M-CSF) and the receptor activator of NF-κB ligand (RANKL). We found that M-CSF stimulates CD68 expression and RANKL alters the apparent molecular weight of CD68 as measured by Western immunoblotting. In addition, we explored the significance of CD68 expression in osteoclasts by generating mice that lack expression of CD68. These mice have increased trabecular bone, and in vitro assessment of CD68−/− osteoclasts revealed that, in the absence of CD68, osteoclasts demonstrate an accumulation of intracellular vesicle-like structures, and do not efficiently resorb bone. These findings demonstrate a role for CD68 in the function of osteoclasts, and future studies will determine the mechanistic nature of the defects seen in CD68−/− osteoclasts.

Notch Signaling Promotes Osteoclast Maturation and Resorptive Activity

The role of Notch signaling in osteoclast differentiation is controversial with conflicting experimental evidence indicating both stimulatory and inhibitory roles. Differences in experimental protocols and in vivo versus in vitro models may explain the discrepancies between studies. In this study, we investigated cell autonomous roles of Notch signaling in osteoclast differentiation and function by altering Notch signaling during osteoclast differentiation using stimulation with immobilized ligands Jagged1 or Delta‐like1 or by suppression with γ‐secretase inhibitor DAPT or transcriptional inhibitor SAHM1. Stimulation of Notch signaling in committed osteoclast precursors resulted in larger osteoclasts with a greater number of nuclei and resorptive activity whereas suppression resulted in smaller osteoclasts with fewer nuclei and suppressed resorptive activity. Conversely, stimulation of Notch signaling in osteoclast precursors prior to induction of osteoclastogenesis resulted in fewer osteoclasts. Our data support a mechanism of context‐specific Notch signaling effects wherein Notch stimulation inhibits commitment to osteoclast differentiation, but enhances the maturation and function of committed precursors. J. Cell. Biochem. 116: 2598–2609, 2015.

Novel effects of Brefeldin A (BFA) in signaling through the insulin receptor (IR) pathway and regulating FoxO1-mediated transcription.

Brefeldin A (BFA) is a fungal metabolite best known for its ability to inhibit activation of ADP-ribosylation factor (Arf) and thereby inhibit secretory traffic. BFA also appears to regulate the trafficking of the GLUT4 glucose transporter by inducing its relocation from intracellular stores to the cell surface. Such redistribution of GLUT4 is normally regulated by insulin-mediated signaling. Hence, we tested whether BFA may intersect with the insulin pathway. We report that BFA causes the activation of the insulin receptor (IR), IRS-1, Akt-2, and AS160 components of the insulin pathway. The response is mediated through phosphoinositol-3-kinase (PI3K) and Akt kinase since the PI3K inhibitor wortmannin and the Akt inhibitors MK2206 and perifosine inhibit the BFA effect. BFA-mediated activation of the insulin pathway results in Akt-mediated phosphorylation of the insulin-responsive transcription factor FoxO1. This leads to nuclear exclusion of FoxO1 and a decrease in transcription of the insulin-responsive gene SIRT-1. Our findings suggest novel effects for BFA in signaling and transcription, and imply that BFA has multiple intracellular targets and can be used to regulate diverse cellular responses that include vesicular trafficking, signaling and transcription.

Intervertebral disc development and disease-related genetic polymorphisms

The intervertebral disc (IVD) comprises a gelatinous inner core (nucleus pulposus; NP) and concentric rings (annulus fibrosus; AF). The NP, an important structure for shock absorption in the vertebrate spinal motion segment, can be traced back to the notochord in ontogenetic lineage. In vertebrates, the notochord undergoes mucinoid changes, and had been considered vestigial until recently. However, observed correlations between IVD degeneration and back pain in humans have renewed interest in the IVD in biomedical fields. Beyond its mechanical contribution to development, the notochord is also an essential signaling center, which coordinates formation of the neural tube and somites. The pertinent signaling molecules, particularly TGF-β and bone morphogenetic proteins (BMPs), continue to play roles in the adult tissues and have been utilized for tissue regeneration. Genetic factors are major determinants of who will develop IVD degeneration and related back pain, and seem to correlate better with disc degeneration and back pain than do external forces on the spine. In summary, the spinal column is a landmark development in evolution. Genes directing the development of the IVD may also contribute to its maintenance, degeneration, and regeneration. Likewise, structural genes as well as genes responsible for maintenance of the structure are related to IVD degeneration. Finally, genes responsible for inflammation may play a dual role in exacerbating degeneration or facilitating repair responses depending on the context.

Expression of Osteoprotegerin from a Replicating Adenovirus Inhibits the Progression of Prostate Cancer Bone Metastases in a Murine Model

Metastatic involvement of the skeleton is a frequent consequence of advanced prostate cancer. These skeletal metastases cause a number of debilitating complications and are refractory to current treatments. New therapeutic options are being explored, including conditionally replicating adenoviruses (CRAds). CRAds are engineered to selectively replicate in and destroy tumor cells and can be ‘armed’ with exogenous transgenes for enhanced potency. We hypothesized that a CRAd armed with osteoprotegerin (OPG), an inhibitor of osteoclastogenesis, would inhibit the progression of prostate cancer bone metastases by directly lysing tumor cells and by reducing osteoclast activity. Although prostate cancer bone metastases are predominantly osteoblastic in nature, increased osteoclast activity is critical for the growth of these lesions. Ad5-Δ24-sOPG-Fc-RGD is a CRAd that carries a fusion of the ligand-binding domains of OPG and the Fc region of human IgG1 in place of the viral E3B genes. To circumvent low tumor cell expression of the native adenoviral receptor, an arginine–glycine–aspartic acid (RGD) peptide insertion within the viral fiber knob allows infection of cells expressing αv integrins. A 24-base pair deletion (Δ24) within viral E1A limits replication to cells with aberrant retinoblastoma cell cycle regulator/tumor suppressor expression. We have confirmed that Ad5-Δ24-sOPG-Fc-RGD replicates within and destroys prostate cancer cells and, in both murine and human coculture models, that infection of prostate cancer cells inhibits osteoclastogenesis in vitro. In a murine model, progression of advanced prostate cancer bone metastases was inhibited by treatment with Ad5-Δ24-sOPG-Fc-RGD but not by an unarmed control CRAd.

Skeletal Involvement in Alagille Syndrome

Alagille syndrome results in a spectrum of skeletal abnormalities in patients. Distinct facies are pathognomonic for the disease, and butterfly vertebrae are common. Patients have reduced bone mass and a propensity to fracture. While some of these abnormalities are developmental, others may be associated with alterations in nutrition (malabsorption) and systemic metabolism during growth. Emerging data demonstrate that Notch signaling and Jagged1 in particular have direct effects on adult bone cells. Activating Notch signaling with Jagged1 in human cells promotes osteoblast differentiation. However, data in genetically modified mice suggests that the regulation of Jagged1-Notch on the skeleton is complex, and Notch signaling may have variable effects depending on the site of the bone and the differentiation status of the target cell. Excitingly, the most recent work in the field demonstrates that delivery of Jagged1 bound to biomaterials promotes bone formation. This has broad implications for treating Alagille syndrome patients with acute fractures as well as treating patients with osteopenia.