The selective degradation of many short-lived proteins in eukaryotic cells is carried out by the ubiquitin system. In this pathway, proteins are targeted for degradation by covalent ligation to ubiquitin, a highly conserved small protein. Ubiquitin-mediated degradation of regulatory proteins plays important roles in the control of numerous processes, including cell-cycle progression, signal transduction, transcriptional regulation, receptor down-regulation, and endocytosis. The ubiquitin system has been implicated in the immune response, development, and programmed cell death. Abnormalities in ubiquitin-mediated processes have been shown to cause pathological conditions, including malignant transformation. In this review we discuss recent information on functions and mechanisms of the ubiquitin system. Since the selectivity of protein degradation is determined mainly at the stage of ligation to ubiquitin, special attention is focused on what we know, and would like to know, about the mode of action of ubiquitin-protein ligation systems and about signals in proteins recognized by these systems.

The Ubiquitin System

Osteoprotegerin Ligand Is a Cytokine that Regulates Osteoclast Differentiation and Activation

Osteoclasts, the multinucleated cells that resorb bone, develop from hematopoietic cells of monocyte/macrophage lineage. Osteoclast-like cells (OCLs) are formed by coculturing spleen cells with osteoblasts or bone marrow stromal cells in the presence of bone-resorbing factors. The cell-to-cell interaction between osteoblasts/stromal cells and osteoclast progenitors is essential for OCL formation. Recently, we purified and molecularly cloned osteoclastogenesis-inhibitory factor (OCIF), which was identical to osteoprotegerin (OPG). OPG/OCIF is a secreted member of the tumor necrosis factor receptor family and inhibits osteoclastogenesis by interrupting the cell-to-cell interaction. Here we report the expression cloning of a ligand for OPG/OCIF from a complementary DNA library of mouse stromal cells. The protein was found to be a member of the membrane-associated tumor necrosis factor ligand family and induced OCL formation from osteoclast progenitors. A genetically engineered soluble form containing the extracellular domain of the protein induced OCL formation from spleen cells in the absence of osteoblasts/stromal cells. OPG/OCIF abolished the OCL formation induced by the protein. Expression of its gene in osteoblasts/stromal cells was up-regulated by bone-resorbing factors. We conclude that the membrane-bound protein is osteoclast differentiation factor (ODF), a long-sought ligand mediating an essential signal to osteoclast progenitors for their differentiation into osteoclasts. ODF was found to be identical to TRANCE/RANKL, which enhances T-cell growth and dendritic-cell function. ODF seems to be an important regulator in not only osteoclastogenesis but also immune system.

https://www.pnas.org/content/pnas/95/7/3597.full.pdf

Osteoclast differentiation factor is a ligand for osteoprotegerin/osteoclastogenesis-inhibitory factor and is identical to TRANCE/RANKL

The tumour-necrosis-factor-family molecule osteoprotegerin ligand (OPGL; also known as TRANCE, RANKL and ODF) has been identified as a potential osteoclast differentiation factor and regulator of interactions between T cells and dendritic cells in vitro. Mice with a disrupted opgl gene show severe osteopetrosis and a defect in tooth eruption, and completely lack osteoclasts as a result of an inability of osteoblasts to support osteoclastogenesis. Although dendritic cells appear normal, opgl-deficient mice exhibit defects in early differentiation of T and B lymphocytes. Surprisingly, opgl-deficient mice lack all lymph nodes but have normal splenic structure and Peyer's patches. Thus OPGL is a new regulator of lymph-node organogenesis and lymphocyte development and is an essential osteoclast differentiation factor in vivo.

OPGL is a key regulator of osteoclastogenesis, lymphocyte development and lymph-node organogenesis

Interleukin 6 (IL-6), promptly and transiently produced in response to infections and tissue injuries, contributes to host defense through the stimulation of acute phase responses, hematopoiesis, and immune reactions. Although its expression is strictly controlled by transcriptional and posttranscriptional mechanisms, dysregulated continual synthesis of IL-6 plays a pathological effect on chronic inflammation and autoimmunity. For this reason, tocilizumab, a humanized anti-IL-6 receptor antibody was developed. Various clinical trials have since shown the exceptional efficacy of tocilizumab, which resulted in its approval for the treatment of rheumatoid arthritis and juvenile idiopathic arthritis. Moreover, tocilizumab is expected to be effective for other intractable immune-mediated diseases. In this context, the mechanism for the continual synthesis of IL-6 needs to be elucidated to facilitate the development of more specific therapeutic approaches and analysis of the pathogenesis of specific diseases.

/pdf/il-6-in-inflammation-immunity-and-disease-4vb9xupl1q.pdf

IL-6 in Inflammation, Immunity, and Disease

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1016/S0014-5793%2897%2901480-4

Oncoprotein MDM2 is a ubiquitin ligase E3 for tumor suppressor p53

Interleukin-6 (IL-6) is a multifunctional cytokine whose circulating levels are under physiological conditions below detection, but whose production is rapidly and strongly induced by several pathological and inflammatory stimuli. IL-6 has been implicated in a number of cell functions connected to immunity and hematopoiesis. Recently, it has been proposed to act as a stimulator of osteoclast formation and activity, in particular following estrogen depletion. The purpose of this study was to gain additional insights into the role of IL-6 during development, as well as in physiological and pathological conditions. We report here that IL-6 deficient mice generated by gene targeting are viable and do not present any evident phenotypic abnormality. However, analysis of bone metabolism revealed a specific bone phenotype. IL-6 deficient female mice have a normal amount of trabecular bone, but higher rates of bone turnover than control littermates. Estrogen deficiency induced by ovariectomy causes in wild type animals a significant loss of bone mass together with an increase in bone turnover rates. Strikingly, ovariectomy does not induce any change in either bone mass or bone remodeling rates in the IL-6 deficient mice. These findings indicate that IL-6 plays an important role in the local regulation of bone turnover and, at least in mice, appears to be essential for the bone loss caused by estrogen deficiency.

Interleukin-6 deficient mice are protected from bone loss caused by estrogen depletion.

It has been postulated that reactive oxygen species (ROS) may act as second messengers leading to nuclear factor (NF)-kappaB activation. This hypothesis is mainly based on the findings that N-acetyl-L-cysteine (NAC) and pyrrolidine dithiocarbamate (PDTC), compounds recognized as potential antioxidants, can inhibit NF-kappaB activation in a wide variety of cell types. Here we reveal that both NAC and PDTC inhibit NF-kappaB activation independently of antioxidative function. NAC selectively blocks tumor necrosis factor (TNF)-induced signaling by lowering the affinity of receptor to TNF. PDTC inhibits the IkappaB-ubiquitin ligase activity in the cell-free system where extracellular stimuli-regulated ROS production does not occur. Furthermore, we present evidence that endogenous ROS produced through Rac/NADPH oxidase do not mediate NF-kappaB signaling, but instead lower the magnitude of its activation.

https://www.embopress.org/doi/pdf/10.1093/emboj/cdg332

Evidence that reactive oxygen species do not mediate NF‐κB activation

Alendronate (ALN), an aminobisphosphonate used in the treatment of osteoporosis, is a potent inhibitor of bone resorption. Its molecular target is still unknown. This study examines the effects of ALN on the activity of osteoclast protein-tyrosine phosphatase (PTP; protein-tyrosine-phosphate phosphohydrolase, EC 3.1.3.48), called PTPepsilon. Using osteoclast-like cells generated by coculturing mouse bone marrow cells with mouse calvaria osteoblasts, we found by molecular cloning and RNA blot hybridization that PTPepsilon is highly expressed in osteoclastic cells. A purified fusion protein of PTPepsilon expressed in bacteria was inhibited by ALN with an IC50 of 2 microM. Other PTP inhibitors--orthovanadate and phenylarsine oxide (PAO)-inhibited PTPepsilon with IC50 values of 0.3 microM and 18 microM, respectively. ALN and another bisphosphonate, etidronate, also inhibited the activities of other bacterially expressed PTPs such as PTPsigma and CD45 (also called leukocyte common antigen). The PTP inhibitors ALN, orthovanadate, and PAO suppressed in vitro formation of multinucleated osteoclasts from osteoclast precursors and in vitro bone resorption by isolated rat osteoclasts (pit formation) with estimated IC50 values of 10 microM, 3 microM, and 0.05 microM, respectively. These findings suggest that tyrosine phosphatase activity plays an important role in osteoclast formation and function and is a putative molecular target of bisphosphonate action.

https://www.pnas.org/content/pnas/93/7/3068.full.pdf

Protein-tyrosine phosphatase activity regulates osteoclast formation and function: inhibition by alendronate

A novel Smt3-specific isopeptidase, SMT3IP1, was cloned using a yeast two-hybrid screen with Smt3b as bait. The clone, named SMT3IP1 (Smt3-specific isopeptidase 1), which bound to Smt3b but not SUMO-1 in the two-hybrid system, was distantly related to budding yeast Saccharomyces cerevisiae Ulp1, human SENP1 or human SUSP1. The catalytic domains in the C-terminal region were very similar, but the N-terminal region was quite different to other enzymes. The cysteine, histidine and asparatic acid residues in the catalytic domains were conserved. SMT3IP1 expressed by the baculovirus-expression system had the ability to cleave SUMO-1 or Smt3b from SUMO-1/RanGAP1 or Smt3b/RanGAP1 conjugates, respectively, and the activity was a little stronger towards the Smt3b conjugate than towards the SUMO-1 conjugate. Furthermore, the enzyme bound more strongly to Smt3a and Smt3b than to SUMO-1 in vitro. The enzyme did not cleave Nedd8 from Nedd8/cullin-1. Nor did it cleave ubiquitin from ubiquitinated p53. SMT3IP1 was localized almost exclusively at the nucleolus during interphase. The N-terminal sequence was responsible for the nucleolar localization of this enzyme. Whether SMT3IP1 functions in the nucleolus or just stays there before it functions in the nucleus, as shown in the case of CDC14 phosphatase, remains to be elucidated.

Hirofumi Tanaka

Papers

Oncoprotein MDM2 is a ubiquitin ligase E3 for tumor suppressor p53

Interleukin-6 deficient mice are protected from bone loss caused by estrogen depletion.

Evidence that reactive oxygen species do not mediate NF‐κB activation

Protein-tyrosine phosphatase activity regulates osteoclast formation and function: inhibition by alendronate

A novel mammalian Smt3-specific isopeptidase 1 (SMT3IP1) localized in the nucleolus at interphase.