Xuezhong Qin

TNF-related weak inducer of apoptosis (TWEAK) is a potent skeletal muscle-wasting cytokine

TWEAK cytokine has been implicated in several biological responses including inflammation, angiogenesis, and osteoclastogenesis. We have investigated the role of TWEAK in regulating skeletal muscle mass. Addition of soluble TWEAK protein to cultured myotubes reduced the mean myotube diameter and enhanced the degradation of specific muscle proteins such as CK and MyHCf. The effect of TWEAK on degradation of MyHCf was stronger than its structural homologue, TNF‐α. TWEAK increased the ubiquitination of MyHCf and the transcript levels of atrogin‐1 and MuRF1 ubiquitin ligases. TWEAK inhibited phosphor‐ylation of Akt kinase and its downstream targets GSK‐3β, FOXO1, mTOR, and p70S6K Furthermore, TWEAK increased the activation of NF‐κB transcription factor in myotubes. Adenoviral‐mediated overexpression of IκBαΔN (a degradation‐resistant mutant of NF‐κB inhibitory protein ΙκBα) in myotubes blocked the TWEAK‐induced degradation of MyHCf. Chronic administration of TWEAK in mice resulted in reduced body and skeletal muscle weight with an associated increase in the activity of ubiquitin‐proteasome system and NF‐κB. Finally, muscle‐specific transgenic overexpression of TWEAK decreased the body and skeletal muscle weight in mice. Collectively, our data suggest that TWEAK induces skeletal muscle atrophy through inhibition of the PI3K/Akt signaling pathway and activation of the ubiquitin‐proteasome and NF‐kB systems.—Dogra, C., Changotra, H., Wedhas, N., Qin, X., Wergedal, J. E., Kumar, A. TNF‐related weak inducer of apoptosis (TWEAK) is a potent skeletal muscle‐wasting cytokine. FASEB J. 21, 1857–1869 (2007)

Structure-function analysis of the human insulin-like growth factor binding protein-4.

To identify the molecular mechanism by which insulin-like growth factor binding protein-4 (IGFBP-4) exerts its inhibitory effects on insulin-like growth factor (IGF) actions, we localized and determined the role of the IGF binding domain in modulating IGF actions in human osteoblasts. Deletion analysis using IGFBP-4 expressed in bacteria revealed that the N-terminal sequence Leu72–Ser91 was essential for IGF binding. The C-terminal fragments (His121–Glu237 or Arg142–Glu237) did not bind to IGF but loss of these regions decreased IGF binding activity. Detailed deletion analysis identified the residues Cys205–Val214as the motif to facilitate IGF binding. Mitogenic studies revealed that an IGFBP-4 mutant (His74 replaced by Pro74) and an N-terminal peptide (N terminus to Thr71) with little IGF binding activity failed to inhibit IGF-II-induced human osteoblast proliferation. An N-terminal peptide (N terminus to Asn182) with reduced IGF binding activity inhibited IGF action but with lower potency. In contrast, an IGFBP-4 mutant (His74 replaced with Ala74) exhibited similar IGF binding activity and potency in inhibiting the activity of IGF-II compared with the wild type. Therefore, the N-terminal sequence (Leu72–Ser91) and the C-terminal sequence (Cys205–Val214) are necessary to form the high affinity IGF binding domain, which is the major structural determinant of the IGFBP-4 function.

Effects of recombinant insulin-like growth factor-binding protein-4 on bone formation parameters in mice.

Insulin-like growth factor (IGF)-binding protein-4 (IGFBP-4), one of the most abundant IGFBPs produced by bone cells, is a potent inhibitor of IGF actions in vitro. To evaluate the modulation of IGF actions on bone formation in vivo by IGFBP-4, we produced intact and fragment (50- to 100-fold reduced IGF affinity) forms of BP-4 and examined their local and systemic effects using biochemical markers. Local administration of IGF-I over the right parietal bone significantly increased bone extract alkaline phosphatase activity; this was completely blocked by an equimolar dose of intact IGFBP-4, but not IGFBP-4 fragment. A single sc administration of IGF-I (2 μg/g BW) significantly increased bone formation markers in both serum and skeletal extracts; surprisingly, so did intact IGFBP-4, but not fragment IGFBP-4. Subcutaneous administration of an equimolar dose of IGFBP-4 along with IGF-I did not significantly block the IGF-I effect. Administration of intact IGFBP-4 significantly increased the serum 50-kDa IGF ...

Tumor Necrosis Factor-α Augments Matrix Metalloproteinase-9 Production in Skeletal Muscle Cells through the Activation of Transforming Growth Factor-β-activated Kinase 1 (TAK1)-dependent Signaling Pathway

We have investigated the effect of tumor necrosis factor-α (TNF-α) on the production of extracellular matrix-degrading proteases in skeletal muscles. Using microarray, quantitative PCR, Western blotting, and zymography, we found that TNF-α drastically increases the production of matrix metalloproteinase (MMP)-9 from C2C12 myotubes. In vivo administration of TNF-α in mice increased the transcript level of MMP-9 in skeletal muscle tissues. Although TNF-α activated all the three MAPKs (i.e. ERK1/2, JNK, and p38), inhibition of ERK1/2 or p38 but not JNK blunted the TNF-α-induced production of MMP-9 from myotubes. Inhibition of Akt also inhibited the TNF-α-induced production of MMP-9. TNF-α increased the activation of transcription factors NF-κB and AP-1 but not SP-1 in myotubes. Overexpression of a dominant negative inhibitor of NF-κB or AP-1 blocked the TNF-α-induced expression of MMP-9 in myotubes. Similarly, point mutations in AP-1- or NF-κB-binding sites in MMP-9 promoter inhibited the TNF-α-induced expression of a reporter gene. TNF-α increased the activity of transforming growth factor-β-activating kinase-1 (TAK1). Furthermore, overexpression of a dominant negative mutant of TAK1 blocked the TNF-α-induced expression of MMP-9 and activation of NF-κB and AP-1. Our results also suggest that TNF-α induces MMP-9 expression in muscle cells through the recruitment of TRAF-2, Fas-associated protein with death domain, and TNF receptor-associated protein with death domain but not NIK or TRAF-6 proteins. We conclude that TAK1-mediated pathways are involved in TNF-α-induced MMP-9 production in skeletal muscle cells.

Studies on the Role of Human Insulin‐like Growth Factor‐II (IGF‐II)‐Dependent IGF Binding Protein (hIGFBP)‐4 Protease in Human Osteoblasts Using Protease‐Resistant IGFBP‐4 Analogs

To characterize the insulin‐like growth factor binding protein‐4 (IGFBP‐4) protease produced by human osteoblasts (hOBs), we localized and determined the role of the proteolytic domains in human IGFBP‐4 (hIGFBP‐4) in modulating IGF‐II actions. N‐terminal amino acid sequence and mass spectrometric analyses of the 6xHis‐tagged IGFBP‐4 proteolytic fragments revealed that Met135‐Lys136 was the only cleavage site recognized by the IGF‐II–dependent IGFBP‐4 protease produced by hOBs. This cleavage site was confirmed by the finding that deletion of His121 to Pro141 blocked proteolysis. However, unexpectedly, deletion of Pro94 to Gln119 containing no cleavage site had no effect on IGF‐II binding activity but blocked proteolysis. Addition of the synthetic peptide corresponding to this region at concentrations of 250 or 1000 molar excess failed to block IGFBP‐4 proteolysis. These data suggest that residues 94–119 may be involved in maintaining the IGFBP‐4 conformation required to expose the cleavage site rather than being involved in direct protease‐substrate binding. To determine the physiological significance of the IGF‐II–dependent IGFBP‐4 protease, we compared the effect of the wild‐type IGFBP‐4 and the protease‐resistant IGFBP‐4 analogs in blocking IGF‐II–induced cell proliferation in normal hOBs, which produce IGFBP‐4 protease, and MG63 cells, which do not produce IGFBP‐4 protease. It was found that protease‐resistant IGFBP‐4 analogs were more potent than the wild‐type protein in inhibiting IGF‐II–induced cell proliferation in hOBs but not in MG63 cells. These data suggest that IGFBP‐4 proteolytic fragments are not biologically active and that IGFBP‐4 protease plays an important role in regulating IGFBP‐4 bioavailability and consequently the mitogenic activity of IGFs in hOBs.

Pregnancy-associated Plasma Protein-A Regulates Myoblast Proliferation and Differentiation through an Insulin-like Growth Factor-dependent Mechanism

Pregnancy-associated plasma protein-A (PAPP-A), a member of the metalloproteinase superfamily, is an important regulator of mammalian growth and development. However, the role of PAPP-A and its mechanism of action in various cellular processes remain unknown. In this study, we have investigated the role of PAPP-A in skeletal myogenesis using C2C12 myoblasts. Recombinant PAPP-A was purified from the conditioned medium of HT1080 cells overexpressing PAPP-A. Treatment of C2C12 myoblasts with PAPP-A increased their proliferation in a dose- and time-dependent manner. Addition of exogenous PAPP-A also increased the myotube formation and the activity of creatine kinase in C2C12 cultures. Transient overexpression of the full-length PAPP-A-(1-1547), but not truncated protease-inactive N-terminal PAPP-A-(1-920) or C-terminal PAPP-A-(1100-1547), significantly enhanced the proliferation of C2C12 myoblasts. In vitro and in situ experiments demonstrated that PAPP-A cleaves insulin-like growth factor-binding protein (IGFBP)-2, but not IGFBP-3, in the conditioned medium of C2C12 myoblasts. Overexpression of PAPP-A led to degradation of the IGFBP-2 produced by C2C12 myoblasts and increased free IGF-I concentrations without affecting total IGF-I concentrations. Addition of protease-resistant IGFBP-4 completely abolished the PAPP-A-induced proliferation of C2C12 myoblasts. Our results demonstrate that 1) PAPP-A increases the proliferation and differentiation of myoblasts, 2) the stimulatory effect of PAPP-A on myogenesis is governed by its proteolytic activity, and 3) PAPP-A promotes skeletal myogenesis by increasing the amount of free IGFs via specific degradation of IGFBP-2 produced by myoblasts.

1,25-Dihydroxyvitamin D3 suppresses TLR8 expression and TLR8-mediated inflammatory responses in monocytes in vitro and experimental autoimmune encephalomyelitis in vivo.

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) suppresses autoimmunity and inflammation; however, the mechanism of its action has not been fully understood. We sought in this study to determine whether the anti-immune/anti-inflammatory action of 1,25(OH)2D3 is in part mediated through an interplay between 1,25(OH)2D3 and toll-like receptor (TLR)7/8 signaling. 1,25(OH)2D3 treatment prior to and/or following experimental autoimmune encephalomyelitis (EAE) induction effectively reduced inflammatory cytokine expression in the spinal cord and ameliorated EAE. These effects were accompanied with a reduction in expression of several TLRs with the most profound effect observed for TLR8. The expression of TLR8 adaptor protein MyD88 was also significantly reduced by 1,25(OH)2D3. To determine the molecular mechanism by which 1,25(OH)2D3 suppresses EAE induction of TLR8 and inflammatory cytokine expression, we evaluated whether 1,25(OH)2D3 can directly inhibit TLR8 signaling and the resulting inflammatory responses in human THP-1 monocytes. 1,25(OH)2D3 treatment not only significantly reduced TLR8 expression but also the expression or activity of MyD88, IRF-4, IRF-7 and NF-kB in monocytes challenged with TLR8 ligands. TLR8 promoter-luciferase reporter assays indicated that 1,25(OH)2D3 decreases TLR8 mRNA level in part via inhibiting TLR8 gene transcription activity. As a result of inhibition on TLR8 signaling cascade at various stages, 1,25(OH)2D3 significantly diminished the TLR8 target gene expression (TNF-α and IL-1β). In summary, our novel findings suggest that TLR8 is a new target of 1,25(OH)2D3 and may mediate the anti-inflammatory action of 1,25(OH)2D3. Our findings also point to a destructive role of TLR8 in EAE and shed lights on pathogenesis of multiple sclerosis.

Targeted Disruption of Ephrin B1 in Cells of Myeloid Lineage Increases Osteoclast Differentiation and Bone Resorption in Mice

Disruption of ephrin B1 in collagen I producing cells in mice results in severe skull defects and reduced bone formation. Because ephrin B1 is also expressed during osteoclast differentiation and because little is known on the role of ephrin B1 reverse signaling in bone resorption, we examined the bone phenotypes in ephrin B1 conditional knockout mice, and studied the function of ephrin B1 reverse signaling on osteoclast differentiation and resorptive activity. Targeted deletion of ephrin B1 gene in myeloid lineage cells resulted in reduced trabecular bone volume, trabecular number and trabecular thickness caused by increased TRAP positive osteoclasts and bone resorption. Histomorphometric analyses found bone formation parameters were not changed in ephrin B1 knockout mice. Treatment of wild-type precursors with clustered soluble EphB2-Fc inhibited RANKL induced formation of multinucleated osteoclasts, and bone resorption pits. The same treatment of ephrin B1 deficient precursors had little effect on osteoclast differentiation and pit formation. Similarly, activation of ephrin B1 reverse signaling by EphB2-Fc treatment led to inhibition of TRAP, cathepsin K and NFATc1 mRNA expression in osteoclasts derived from wild-type mice but not conditional knockout mice. Immunoprecipitation with NHERF1 antibody revealed ephrin B1 interacted with NHERF1 in differentiated osteoclasts. Treatment of osteoclasts with exogenous EphB2-Fc resulted in reduced phosphorylation of ezrin/radixin/moesin. We conclude that myeloid lineage produced ephrin B1 is a negative regulator of bone resorption in vivo, and that activation of ephrin B1 reverse signaling inhibits osteoclast differentiation in vitro in part via a mechanism that involves inhibition of NFATc1 expression and modulation of phosphorylation status of ezrin/radixin/moesin.

Differential regulation of pregnancy associated plasma protein (PAPP): A during pregnancy in human and mouse

UNLABELLED Serum IGFBP-4 proteolytic activity increases dramatically during human pregnancy and is mainly attributed to the pregnancy-associated plasma protein-A (PAPP-A). To understand the regulation and actions of PAPP-A in vivo, we evaluated the utility of a mouse model system. Serum from day-9 and day-17 pregnant mice and age-matched controls was tested for IGFBP-4 proteolytic activity using recombinant mouse IGFBP-4 as the substrate. Surprisingly, IGFBP-4 proteolytic activity in mouse pregnancy serum (mPS) was not significantly different from that of non-pregnancy serum (mNPS). Addition of IGF-II to mPS or mNPS at a dose sufficient to increase IGFBP-4 proteolysis by human PS failed to enhance IGFBP-4 proteolysis. PAPP-A neutralization antibody did not inhibit IGFBP-4 proteolysis by mPS or mNPS, but completely blocked IGFBP-4 proteolytic activity in human PS (hPS, mouse osteoblast conditioned medium, and mouse amniotic fluid). To determine whether the lack of PAPP-A activity in mPS was due to low expression of PAPP-A in the placenta, we cloned a mouse genomic DNA, which contained 1 kb of the entire exon 2 coding sequence and 2.5 kb of the flanking intron sequences. The exon 2-coded mouse and human PAPP-A shared 86% amino acid sequence identity. RT-PCR analysis revealed that the PAPP-A mRNA level in mouse placenta was lower compared to that in human placenta by at least two orders of magnitude. PAPP-A expression was also lower in mouse placenta compared to those in mouse kidney, osteoblasts, and bone marrow stromal cells. CONCLUSIONS (1) Serum IGFBP-4 proteolytic activity is differentially regulated by pregnancy in human and mouse. (2) The lack of an increase in serum IGFBP-4 proteolytic activity during mouse pregnancy is due to the low level of PAPP-A expression in the placenta.

Inactivation of insulin-like-growth factors diminished the anabolic effects of pregnancy-associated plasma protein-A (PAPP-A) on bone in mice

In vivo studies have provided ubiquitous evidence that pregnancy-associated plasma protein-A (PAPP-A) functions as a potent anabolic factor. While some evidence supports the prediction that increasing IGF bioavailability contributes to the anabolic effects of PAPP-A, definitive evidence has been lacking. This important issue has been addressed in this study using a unique mouse model in which PAPP-A was overexpressed in bone either alone or together with a protease-resistant IGFBP-4 analog (PRBP-4) which serves as an IGF inhibitor. PAPP-A transgenic mice exhibited a 25% increase in skull bone mineral density (BMD) whereas PRBP-4 transgenic mice showed a 20-25% decrease in this parameter at an age of 3months. Femur/tibia size-related parameters were significantly increased in PAPP-A transgenic mice but decreased in PRBP-4 transgenic mice. This data clearly demonstrates that PAPP-A transgenic mice exhibit opposite phenotypes in both flat bone and long bone compared to PRBP-4 transgenic mice which have reduced IGF bioavailability in bone. Importantly, PRBP-4 and PRBP-4/PAPP-A double transgenic mice shared essentially identical phenotypes in both flat and long bones. Calvarial thickness, skull BMD and long bone parameters were reduced to similar degrees in PRBP-4 and PRBP-4/PAPP-A transgenic mice relative to wild-type littermates. Our findings provide compelling evidence that PAPP-A increases bone formation primarily by increasing IGF bioavailability and that other alternative pathways may play a negligible role in mediating the anabolic effect of PAPPA in bone. This clear definition of PAPP-As mechanism of action is critical for future translational studies on the therapeutic application of PAPP-A.