Osteoactivin fragments produced by ectodomain shedding induce MMP-3 expression via ERK pathway in mouse NIH-3T3 fibroblasts.

Intact osteoactivin, a novel type I membrane glycoprotein, were shed at a dibasic motif in the juxtamembrane region in C2C12 myoblasts. Extracellular fragments were secreted into the culture media by a putative metalloprotease. Extracellular fragments of osteoactivin, but not control protein, induced matrix metalloprotease-3 (MMP-3) expression in NIH-3T3 fibroblasts. Epidermal growth factor (ERK) kinase inhibitors inhibited the osteoactivin-mediated MMP-3 expression, whereas the extracellular fragment of osteoactivin activated ERK1/2 and p38 in the mitogen-activated protein kinase pathway. Our results suggest that the extracellular fragments of osteoactivin produced by shedding act as a growth factor to induce MMP-3 expression via the ERK pathway in fibroblasts.

Overexpression of osteoactivin protects skeletal muscle from severe degeneration caused by long-term denervation in mice.

We have previously shown that osteoactivin, a type I membrane glycoprotein expressed in myofibers, upregulated expression of matrix metalloprotease (MMP)-3 and MMP-9 in fibroblasts infiltrated denervated skeletal muscle in mice. To address whether osteoactivin-mediated increase in MMPs in skeletal muscle is useful for regeneration of denervated skeletal muscle, we subjected osteoactivin-transgenic mice to long-term denervation for 70 or 90 days. Long-term denervation caused severe degeneration of myofibers and fibrosis in skeletal muscle of wild-type mice. However, overexpression of osteoactivin protected skeletal muscle from such changes. Infiltration of fibroblast-like cells and collagen deposition were sustained at low levels after long-term denervation in skeletal muscle of osteoactivin-transgenic mice. This cytoprotective effect of osteoactivin was supported by the expression of regeneration/degeneration-associated genes in the gastrocnemius muscle during denervation. Denervation significantly upregulated the expression of anti-fibrotic genes, such as glypican-1 and decorin-1, in the gastrocnemius muscle of osteoactivin-transgenic mice, compared with wild-type mice. In contrast, overexpression of osteoactivin caused a significant reduction in denervation-induced expression of elongation factor 1A-1, an indicator for the persistence of degenerated cells. Our results suggest that an osteoactivin-mediated increase in MMPs in skeletal muscle might be useful for protecting injured muscle from fibrosis, leading to full regeneration after denervation.

Deficiency of Cbl-b gene enhances infiltration and activation of macrophages in adipose tissue and causes peripheral insulin resistance in mice.

OBJECTIVE: c-Cbl plays an important role in whole-body fuel homeostasis by regulating insulin action. In the present study, we examined the role of Cbl-b, another member of the Cbl family, in insulin action. RESEARCH DESIGN AND METHODS: C57BL/6 (Cbl-b(+/+)) or Cbl-b-deficient (Cbl-b(-/-)) mice were subjected to insulin and glucose tolerance tests and a hyperinsulinemic-euglycemic clamp test. Infiltration of macrophages into white adipose tissue (WAT) was assessed by immunohistochemistry and flow cytometry. We examined macrophage activation using co-cultures of 3T3-L1 adipocytes and peritoneal macrophages. RESULTS: Elderly Cbl-b(-/-) mice developed glucose intolerance and peripheral insulin resistance; serum insulin concentrations after a glucose challenge were always higher in elderly Cbl-b(-/-) mice than age-matched Cbl-b(+/+) mice. Deficiency of the Cbl-b gene significantly decreased the uptake of 2-deoxyglucose into WAT and glucose infusion rate, whereas fatty liver was apparent in elderly Cbl-b(-/-) mice. Cbl-b deficiency was associated with infiltration of macrophages into the WAT and expression of cytokines, such as tumor necrosis factor-alpha, interleukin-6, and monocyte chemoattractant protein (MCP)-1. Co-culture of Cbl-b(-/-) macrophages with 3T3-L1 adipocytes induced leptin expression and dephosphorylation of insulin receptor substrate 1, leading to impaired glucose uptake in adipocytes. Furthermore, Vav1, a key factor in macrophage activation, was highly phosphorylated in peritoneal Cbl-b(-/-) macrophages compared with Cbl-b(+/+) macrophages. Treatment with a neutralizing anti-MCP-1 antibody improved peripheral insulin resistance and macrophage infiltration into WAT in elderly Cbl-b(-/-) mice. CONCLUSIONS: Cbl-b is a negative regulator of macrophage infiltration and activation, and macrophage activation by Cbl-b deficiency contributes to the peripheral insulin resistance and glucose intolerance via cytokines secreted from macrophages.

Soy protein diet prevents hypermethioninemia caused by portacaval shunt in rats.

In hepatic disorders, abnormal plasma amino acid profiles are observed. In this study, we examined whether soy protein isolate (SPI) improved plasma methionine concentration in the model animals. Portacaval shunt (PCS) increased alanine aminotransferase (ALT) activity and methionine concentration in blood of rats fed a 40% casein diet supplemented with 0.6% methionine (casein-M diet). A 40% SPI diet supplemented with 1.28% methionine (SPI-M diet), which contained the same amount of methionine as that in 40% casein-M diet, normalized plasma ALT activity and methionine level in PCS rats. These effects of a SPI diet may be due to its amino acid composition, since an amino acid mixture diet mimicking a 40% SPI-M diet was also effective to hypermethioninemia of PCS rats. To find key enzymes for the beneficial effect of soy protein, we examined effects of a 40% SPI-M or casein-M diet on the activities of three methionine-metabolizing enzymes in liver of PCS rats. A SPI-M diet stimulated only the activity of cystathionine gamma-lyase, compared with a casein-M diet. A SPI diet has a preventive effect on hypermethioninemia, at least in part, by stimulating cystathionine gamma-lyase activity in liver and may be used for nutritional management of liver disorders with hypermethioninemia.

Ubiquitin ligase gene expression in healthy volunteers with 20-day bedrest.

In animal models, several ubiquitin ligases play an important role in skeletal muscle atrophy caused by unloading. In this study we examined protein ubiquitination and ubiquitin ligase gene expression in quadriceps femoris muscle from healthy volunteers after 20-day bedrest to clarify ubiquitin-dependent proteolysis in human muscles after unloading. During bedrest, thickness and cross-sectional area of the quadriceps femoris muscle decreased significantly by 4.6% and 3.7%, respectively. Ubiquitinated proteins accumulated in these atrophied human muscles. A real-time reverse transcription-polymerase chain reaction system showed that bedrest significantly upregulated expression of two ubiquitin ligase genes, Cbl-b and atrogin-1. We also performed DNA microarray analysis to examine comprehensive gene expression in the atrophied muscle. Bedrest mainly suppressed the expression of muscle genes associated with control of gene expression in skeletal muscle. Our results suggest that, in humans, Cbl-b- or atrogin-1-mediated ubiquitination plays an important role in unloading-induced muscle atrophy, and that unloading stress may preferentially inhibit transcriptional responses in skeletal muscle.

Ubiquitin ligase Cbl-b downregulates bone formation through suppression of IGF-I signaling in osteoblasts during denervation.

UNLABELLED: Unloading can prevent bone formation by osteoblasts. To study this mechanism, we focused on a ubiquitin ligase, Cbl-b, which was highly expressed in osteoblastic cells during denervation. Our results suggest that Cbl-b may mediate denervation-induced osteopenia by inhibiting IGF-I signaling in osteoblasts. INTRODUCTION: Unloading, such as denervation (sciatic neurectomy) and spaceflight, suppresses bone formation by osteoblasts, leading to osteopenia. The resistance of osteoblasts to growth factors contributes to such unloading-mediated osteopenia. However, a detailed mechanism of this resistance is unknown. We first found that a RING-type ubiquitin ligase, Cbl-b, was highly expressed in osteoblastic cells after sciatic neurectomy in mice. In this study, we reasoned that Cbl-b played an important role in the resistance of osteoblasts to IGF-I. MATERIALS AND METHODS: Cbl-b-deficient (Cbl-b(-/-)) or wildtype (Cbl-b(+/+)) mice were subjected to sciatic neurectomy. Bone formation in these mice was assessed by calcein labeling and histomorphometric analyses. We examined IGF-I signaling molecules in femora of these mice by Western blot and immunohistochemical analyses. We also examined the mitogenic response of Cbl-b-overexpressing or -deficient osteoblastic cells to various growth factors. RESULTS: In Cbl-b(+/+) mice, denervation decreased femur mass and bone formation, whereas it increased the expression of Cbl-b protein in osteoprogenitor cells and in osteocalcin-positive cells (osteoblastic cells) in hindlimb bone. In contrast, in Cbl-b(-/-) mice, bone mass and bone formation were sustained during denervation. Denervation inhibited the mitogenic response of osteoprogenitor cells most significantly to IGF-I. Therefore, we focused on Cbl-b-mediated modification of IGF-I signaling. Denervation decreased the amounts of insulin receptor substrate-1 (IRS-1), phosphatidly inositol 3-phosphate kinase (PI3K), and Akt-1 proteins in femora of Cbl-b(+/+) mice, whereas the amounts of these IGF-I signaling molecules in femora of Cbl-b(-/-) mice were constant after denervation. On a cellular level, primary osteoblastic cells from Cbl-b(-/-) mice were more stimulated to proliferate by IGF-I treatment compared with those from Cbl-b(+/+) mice. Furthermore, overexpression of Cbl-b increased ubiquitination and degradation of IRS-1 in primary Cbl-b(-/-) osteoblastic cells, leading to their impaired mitogenic response to IGF-I. CONCLUSIONS: These results suggest that Cbl-b induces resistance of osteoblasts to IGF-I during denervation by increasing IRS-1 degradation and that Cbl-b-mediated modification of IGF-I signaling may contribute to decreased bone formation during denervation.

Identification of mono-ubiquitinated LDH-A in skeletal muscle cells exposed to oxidative stress.

We previously reported that oxidative stress is associated with unloading-mediated ubiquitination of muscle proteins. To further elucidate the involvement of oxidative stress in ubiquitination, we examined the ubiquitination profile in rat myoblastic L6 cells after treatment with hydrogen peroxide. Hydrogen peroxide induced many ubiquitinated proteins with low molecular masses (less than 60 kDa) as well as high molecular masses (more than 160 kDa). Among them, a 42-kDa-ubiquitinated protein was abundantly accumulated and immediately disappeared after the treatment. Microsequencing revealed that the 42-kDa-protein was identical to the mono-ubiquitinated form of rat lactate dehydrogenase A (LDH-A), and we confirmed that hydrogen peroxide induced the mono-ubiquitination of LDH-A in COS7 cells overexpressing LDH-A and ubiquitin. Under unloading conditions, such as tail-suspension and spaceflight, mono-ubiquitinated LDH was accumulated in gastrocnemius muscle. Interestingly, E-64-d plus pepstatin, lysosomal protease inhibitors, further accumulated mono-ubiquitinated LDH-A in the cells after treatment with hydrogen peroxide, while they did not affect the amount of poly-ubiquitinated LDH. In contrast, epoxomicin, a potent proteasome inhibitor, did not change the amount of mono-ubiquitinated LDH-A in L6 cells treated with hydrogen peroxide, although it significantly increased the amount of poly-ubiquitinated LDH. Our results suggest that oxidative stress induces not only poly-ubiquitination but also mono-ubiquitination of LDH-A, which may be involved in its lysosomal degradation during unloading.

Short-term hypergravity does not affect protein-ubiquitination and proliferation in rat L6 myoblastic cells.

We previously reported that spaceflight (STS-90) and tail-suspension stimulated muscle protein ubiquitination and accumulated the degradation fragments. However, in space experiments the side-effects of hypergravity on samples are inevitable during the launch of a space shuttle into space or the reentry. To examine whether hypergravity also caused protein-ubiquitination in skeletal muscle cells, we exposed rat myoblastic L6 cells to various hypergravity conditions. Immunoblot analysis showed that the centrifugation at 2, 3, 30 or 100 G for 10 min did not increase the amount of ubiquitinated proteins in L6 cells, whereas the centrifugation at 100 G for 1 or 2 hrs significantly induced the protein-ubiquitination. In contrast, heat shock protein 70 (HSP70), another stress-responsive protein, in L6 cells was accumulated only by centrifugation at 100 G for more than 10 min. Short-term (10 min) hypergravity including 3 or 100 G did not affect the proliferation and morphological changes in L6 cells. Our present results suggest that the ubiquitination of muscle proteins is less sensitive to hypergravity than the induction of HSP70, and that the effect of hypergravity on protein-ubiquitination and proliferation of skeletal muscle cells may be negligible, as far as its duration is short-term.

Osteoactivin upregulates expression of MMP-3 and MMP-9 in fibroblasts infiltrated into denervated skeletal muscle in mice.

In this study, we examined pathophysiological roles of osteoactivin, a functionally unknown type I membrane glycoprotein, in mouse skeletal muscle atrophied by denervation (sciatic neurectomy). Denervation increased the amounts of osteoactivin, vimentin, matrix metalloproteinase-3 (MMP-3), and MMP-9 in mouse gastrocnemius muscle. Interestingly, immunohistochemical analysis revealed that vimentin, MMP-3, and MMP-9 were mainly present in fibroblast-like cells infiltrated into denervated mouse gastrocnemius muscle, whereas osteoactivin was expressed in the sarcolemma of myofibers adjacent to the fibroblast-like cells. On the basis of these findings, we reasoned that osteoactivin in myocytes was involved in activation of the infiltrated fibroblasts. To address this issue, we examined effects of osteoactivin on expression of MMPs in fibroblasts in vitro and in vivo. Overexpression of osteoactivin in NIH-3T3 fibroblasts induced expression of MMP-3, but not in mouse C(2)C(12) myoblasts, indicating that osteoactivin might functionally target fibroblasts. Treatment with recombinant mouse osteoactivin increased the amounts of collagen type I, MMP-3, and MMP-9 in mouse NIH-3T3 fibroblasts. The upregulated expression of these fibroblast marker proteins was significantly inhibited by heparin, but not by an integrin inhibitor, indicating that a heparin-binding motif in the extracellular domain might be an active site of osteoactivin. In osteoactivin-transgenic mice, denervation further enhanced expression of MMP-3 and MMP-9 in fibroblasts infiltrated into gastrocnemius muscle, compared with wild-type mice. Our present results suggest that osteoactivin might function as an activator for fibroblasts infiltrated into denervated skeletal muscles and play an important role in regulating degeneration/regeneration of extracellular matrix.