Daisuke Hoshino

Signaling inputs to invadopodia and podosomes

Summary Remodeling of extracellular matrix (ECM) is a fundamental cell property that allows cells to alter their microenvironment and move through tissues. Invadopodia and podosomes are subcellular actin-rich structures that are specialized for matrix degradation and are formed by cancer and normal cells, respectively. Although initial studies focused on defining the core machinery of these two structures, recent studies have identified inputs from both growth factor and adhesion signaling as crucial for invasive activity. This Commentary will outline the current knowledge on the upstream signaling inputs to invadopodia and podosomes and their role in governing distinct stages of these invasive structures. We discuss invadopodia and podosomes as adhesion structures and highlight new data showing that invadopodia-associated adhesion rings promote the maturation of already-formed invadopodia. We present a model in which growth factor stimulation leads to phosphoinositide 3-kinase (PI3K) activity and formation of invadopodia, whereas adhesion signaling promotes exocytosis of proteinases at invadopodia.

Adhesion rings surround invadopodia and promote maturation

Summary Invasion and metastasis are aggressive cancer phenotypes that are highly related to the ability of cancer cells to degrade extracellular matrix (ECM). At the cellular level, specialized actin-rich structures called invadopodia mediate focal matrix degradation by serving as exocytic sites for ECM-degrading proteinases. Adhesion signaling is likely to be a critical regulatory input to invadopodia, but the mechanism and location of such adhesion signaling events are poorly understood. Here, we report that adhesion rings surround invadopodia shortly after formation and correlate strongly with invadopodium activity on a cell-by-cell basis. By contrast, there was little correlation of focal adhesion number or size with cellular invadopodium activity. Prevention of adhesion ring formation by inhibition of RGD-binding integrins or knockdown (KD) of integrin-linked kinase (ILK) reduced the number of ECM-degrading invadopodia and reduced recruitment of IQGAP to invadopodium actin puncta. Furthermore, live cell imaging revealed that the rate of extracellular MT1-MMP accumulation at invadopodia was greatly reduced in both integrin-inhibited and ILK-KD cells. Conversely, KD of MT1-MMP reduced invadopodium activity and dynamics but not the number of adhesion-ringed invadopodia. These results suggest a model in which adhesion rings are recruited to invadopodia shortly after formation and promote invadopodium maturation by enhancing proteinase secretion. Since adhesion rings are a defining characteristic of podosomes, similar structures formed by normal cells, our data also suggest further similarities between invadopodia and podosomes.

Network analysis of the focal adhesion to invadopodia transition identifies a PI3K-PKCα invasive signaling axis.

The activity of the lipid kinase PI3K governs whether the protein kinase PKCα promotes invasive behavior of cancer cells. Becoming Invasive Invasive and metastatic cancer cells form cellular protrusions called invadopodia that can degrade the extracellular matrix. Hoshino et al. integrated data from head and neck carcinomas with network analysis of invadopodia and focal adhesions, cellular structures that contain many of the same components as invadopodia but have decreased ability to degrade the extracellular matrix. They identified phosphatidylinositol 3-kinase (PI3K) and protein kinase C α (PKCα) as key determinants in the formation of invadopodia. The formation of invadopodia was enhanced by PKCα in cells with wild-type PI3K but was inhibited by PKCα in cells with enhanced PI3K activity (due to expression of components of the PI3K pathway with cancer-associated mutations). These results suggested that PKCα participated in a negative feedback loop that limited the activity of PI3K and thus cellular invasiveness, which the authors confirmed. The combination of high PI3K activity with low PKCα activity correlated with increased number of invadopodia in cell lines derived from head and neck carcinoma, breast cancer, or melanoma. This PI3K-high and PKCα-low signaling state may be useful as a biomarker for cancer aggressiveness. In cancer, deregulated signaling can produce an invasive cellular phenotype. We modeled the invasive transition as a theoretical switch between two cytoskeletal structures: focal adhesions and extracellular matrix–degrading invadopodia. We constructed molecular interaction networks of each structure and identified upstream regulatory hubs through computational analyses. We compared these regulatory hubs to the status of signaling components from head and neck carcinomas, which led us to analyze phosphatidylinositol 3-kinase (PI3K) and protein kinase C α (PKCα). Consistent with previous studies, PI3K activity promoted both the formation and the activity of invadopodia. We found that PI3K induction of invadopodia was increased by overexpression of SH2 (Src homology 2) domain–containing inositol 5′-phosphatase 2 (SHIP2), which converts the phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] that is produced by PI3K activity to phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2], which is believed to promote invadopodia formation. Knockdown of PKCα had divergent effects on invadopodia formation, depending on the status of PI3K. Loss of PKCα inhibited invadopodia formation in cells with wild-type PI3K pathway status. Conversely, in cells with constitutively active PI3K (through activating PI3K mutants or lacking the endogenous opposing enzyme PTEN), PKCα knockdown increased invadopodia formation. Mechanistic studies revealed a negative feedback loop from PKCα that dampened PI3K activity and invasive behavior in cells with genetic hyperactivation of the PI3K pathway. These studies demonstrated the potential of network modeling as a discovery tool and identified PI3K and PKCα as interacting regulators of invasive behavior.

Establishment and Validation of Computational Model for MT1-MMP Dependent ECM Degradation and Intervention Strategies

MT1-MMP is a potent invasion-promoting membrane protease employed by aggressive cancer cells. MT1-MMP localizes preferentially at membrane protrusions called invadopodia where it plays a central role in degradation of the surrounding extracellular matrix (ECM). Previous reports suggested a role for a continuous supply of MT1-MMP in ECM degradation. However, the turnover rate of MT1-MMP and the extent to which the turnover contributes to the ECM degradation at invadopodia have not been clarified. To approach this problem, we first performed FRAP (Fluorescence Recovery after Photobleaching) experiments with fluorescence-tagged MT1-MMP focusing on a single invadopodium and found very rapid recovery in FRAP signals, approximated by double-exponential plots with time constants of 26 s and 259 s. The recovery depended primarily on vesicle transport, but negligibly on lateral diffusion. Next we constructed a computational model employing the observed kinetics of the FRAP experiments. The simulations successfully reproduced our FRAP experiments. Next we inhibited the vesicle transport both experimentally, and in simulation. Addition of drugs inhibiting vesicle transport blocked ECM degradation experimentally, and the simulation showed no appreciable ECM degradation under conditions inhibiting vesicle transport. In addition, the degree of the reduction in ECM degradation depended on the degree of the reduction in the MT1-MMP turnover. Thus, our experiments and simulations have established the role of the rapid turnover of MT1-MMP in ECM degradation at invadopodia. Furthermore, our simulations suggested synergetic contributions of proteolytic activity and the MT1-MMP turnover to ECM degradation because there was a nonlinear and marked reduction in ECM degradation if both factors were reduced simultaneously. Thus our computational model provides a new in silico tool to design and evaluate intervention strategies in cancer cell invasion.

High throughput analysis of proteins associating with a proinvasive MT1-MMP in human malignant melanoma A375 cells

Membrane‐type 1 matrix metalloproteinase (MT1‐MMP), a powerful modulator of the pericellular environment, promotes migration, invasion, and proliferation of cells. To perform its potent proteolytic activity in a controlled manner, MT1‐MMP has to be regulated precisely. However, our knowledge about substrates and regulatory proteins is still very limited. In this study we identify a catalog of proteins that directly or indirectly interact with MT1‐MMP. We expressed a FLAG‐tagged MT1‐MMP stably in human malignant melanoma A375 cells. We prepared cell lysate using Brij98 and MT1‐MMP was affinity purified together with associating proteins using an anti‐FLAG antibody. A distinct set of membrane proteins was found to copurify with MT1‐MMP when biotin‐labeled proteins were monitored. The proteins were analyzed with an integrated system composed of nano‐flow liquid chromatography and tandem mass spectrometry. We identified 158 proteins including several previously reported to bind MT1‐MMP, although most had not previously been identified. Six of these membrane proteins, including one previously shown to interact with MT1‐MMP, were co‐expressed with MT1‐MMP in HT1080 cells. Five of the latter were found to associate with MT1‐MMP in an immunoprecipitation assay. Immunostaining of cells expressing each of these test proteins revealed that one colocalized with MT1‐MMP at the ruffling membrane and the other at the perinuclear vesicles. In contrast, another protein which did not coprecipitate with MT1‐MMP showed no colocalization. Recombinant MT1‐MMP cleaved two of the tested proteins at least in vitro. Thus, we provide a valuable resource to identify substrates and regulators of MT1‐MMP in tumor cells. (Cancer Sci 2009; 100: 1284–1290)

High-intensity interval training increases intrinsic rates of mitochondrial fatty acid oxidation in rat red and white skeletal muscle

High-intensity interval training (HIIT) can increase mitochondrial volume in skeletal muscle. However, it is unclear whether HIIT alters the intrinsic capacity of mitochondrial fatty acid oxidation, or whether such changes are associated with changes in mitochondrial FAT/CD36, a regulator of fatty acid oxidation, or with reciprocal changes in the nuclear receptor coactivator (peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1α)) and the corepressor (receptor-interacting protein 140 (RIP140)). We examined whether HIIT alters fatty acid oxidation rates in the isolated subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria of red and white skeletal muscle and (or) induces changes in muscle PGC-1α and RIP140 proteins and mitochondrial FAT/CD36 protein content. Rats were divided into untrained or HIIT-trained groups. HIIT animals performed 10 bouts of 1-min high-intensity treadmill running (30-55 m·min(-1)), separated by 2 min of rest, for 5 days a week for 4 weeks. As expected, after the training period, HIIT increased mitochondrial enzymes (citrate synthase, COXIV, and β-hydroxyacyl CoA dehydrogenase) in red and white muscle, indicating that muscle mitochondrial volume had increased. HIIT also increased the rates of palmitate oxidation in mitochondria of red (37% for SS and 19% for IMF) and white (36% for SS and 12% for IMF) muscle. No changes occurred in SS and IMF mitochondrial FAT/CD36 proteins, despite increasing FAT/CD36 at the whole-muscle level (27% for red and 22% for white). Concurrently, muscle PGC-1α protein was increased in red (22%) and white (16%) muscle, but RIP140 was not altered. These results indicate that increases in SS and IMF mitochondrial fatty acid oxidation induced by HIIT are accompanied by an increase in PGC-1α, but not RIP140 or FAT/CD36.

Postexercise whole body heat stress additively enhances endurance training-induced mitochondrial adaptations in mouse skeletal muscle

A recent study demonstrated that heat stress induces mitochondrial biogenesis in C2C12 myotubes, thereby implying that heat stress may be an effective treatment to enhance endurance training-induced mitochondrial adaptations in skeletal muscle. However, whether heat stress actually induces mitochondrial adaptations in skeletal muscle in vivo is unclear. In the present study, we report the novel findings that 1) whole body heat stress produced by exposure of ICR mice to a hot environment (40°C, 30 min/day, 5 days/wk, 3 wk) induced mitochondrial adaptations such as increased mitochondrial enzyme activity (citrate synthase and 3-hydroxyacyl CoA dehydrogenase) and respiratory chain protein content (complexes I-V) in skeletal muscle in vivo and 2) postexercise whole body heat stress additively enhanced endurance training-induced mitochondrial adaptations (treadmill running, 25 m/min, 30 min/day, 5 days/wk, 3 wk). Moreover, to determine the candidate mechanisms underlying mitochondrial adaptations, we investigated the acute effects of postexercise whole body heat stress on the phosphorylation status of cellular signaling cascades that subsequently induce mitochondrial gene transcription. We found that whole body heat stress boosted the endurance exercise-induced phosphorylation of p38 MAPK, increased the phosphorylation status of p70S6K, a biomarker of mammalian target of rapamycin complex 1 activity, and unexpectedly dephosphorylated AMP-activated protein kinase and its downstream target acetyl-CoA carboxylase in skeletal muscle. Our present observations suggest that heat stress can act as an effective postexercise treatment. Heat stress treatment appeared to be clinically beneficial for people who have difficulty participating in sufficient exercise training, such as the elderly, injured athletes, and patients.

Daily heat stress treatment rescues denervation-activated mitochondrial clearance and atrophy in skeletal muscle

Traumatic nerve injury or nerve disease leads to denervation and severe muscle atrophy. Recent evidence shows that mitochondrial loss could be a key mediator of skeletal muscle atrophy. Here, we show that daily heat stress treatment rescues denervation‐induced loss of mitochondria and concomitant muscle atrophy. We also found that denervation‐activated autophagy‐dependent mitochondrial clearance (mitophagy) was suppressed by daily heat stress treatment. The molecular basis of this observation is explained by our results showing that heat stress treatment attenuates the increase of key proteins that regulate the tagging step for mitochondrial clearance and the intermediate step of autophagosome formation in denervated muscle. These findings contribute to the better understanding of mitochondrial quality control in denervated muscle from a translational perspective and provide a mechanism behind the attenuation of muscle wasting by heat stress.

Effects of decreased lactate accumulation after dichloroacetate administration on exercise training–induced mitochondrial adaptations in mouse skeletal muscle

Recent studies suggested that lactate accumulation can be a signal for mitochondrial biogenesis in skeletal muscle. We investigated whether reductions in lactate concentrations in response to dichloroacetate (DCA), an activator of pyruvate dehydrogenase, attenuate mitochondrial adaptations after exercise training in mice. We first confirmed that DCA administration (200 mg/kg BW by i.p. injection) 10 min before exercise decreased muscle and blood lactate concentrations after high‐intensity interval exercise (10 bouts of 1 min treadmill running at 40 m/min with a 1 min rest). At the same time, exercise‐induced signal cascades did not change by pre‐exercise DCA administration. These results suggested that DCA administration affected only lactate concentrations after exercise. We next examined the effects of acute DCA administration on mRNA expressions involved with mitochondrial biogenesis after same high‐intensity interval exercise and the effects of chronic DCA administration on mitochondrial adaptations after high‐intensity interval training (increasing intensity from 38 to 43 m/min by the end of training period). Acute DCA administration did not change most of the exercise‐induced mRNA upregulation. These data suggest that lactate reductions by DCA administration did not affect transcriptional activation after high‐intensity interval exercise. However, chronic DCA administration attenuated, in part, mitochondrial adaptations such as training‐induced increasing rates of citrate synthase (P = 0.06), β‐hydroxyacyl CoA dehydrogenase activity (P < 0.05), cytochrome c oxidase IV (P < 0.05) and a fatty acid transporter, fatty acid translocase/CD36 (P < 0.05), proteins after exercise training. These results suggest that lactate accumulation during high‐intensity interval exercise may be associated with mitochondrial adaptations after chronic exercise training.

Effect of growth on monocarboxylate transporters and indicators of energy metabolism in the gluteus medius muscle of Thoroughbreds

OBJECTIVE To examine the changes in monocarboxylate transporter (MCT) 1 and MCT4 content and in indicators of energy metabolism in the gluteus medius muscle (GMM) of Thoroughbreds during growth. ANIMALS 6 Thoroughbreds (3 males and 3 females). PROCEDURES Samples of GMM were obtained when horses were 2, 6, 12, and 24 months old. Muscle proteins were separated via SDS-PAGE; amounts of MCT1 and MCT4 and peroxisome proliferator-activated receptor-γ coactivator-1α content were determined by use of western blotting. Muscle activities of phosphofructokinase and citrate synthase were measured biochemically; lactate dehydrogenase isoenzymes were separated by agarose gel electrophoresis and quantified. RESULTS Compared with findings when horses were 2 months old, MCT1 protein content in GMM samples obtained when the horses were 24 months old was significantly higher; however, MCT4 protein content remained unchanged throughout the study period. Peroxisome proliferator-activated receptor-γ coactivator-1α content was significantly increased at 24 months of age and citrate synthase activity was increased at 6 and 24 months of age, compared with findings at 2 months. Phosphofructokinase activity remained unaltered during growth. The percentage contributions of lactate dehydrogenase 1 and 2 isoenzymes to the total amount of all 5 isoenzymes at 12 and 24 months of age were significantly higher than those at 2 months of age. CONCLUSIONS AND CLINICAL RELEVANCE Changes in protein contents of MCTs and the lactate dehydrogenase isoenzyme profile in GMM samples suggested that lactate usage capacity increases with growth and is accompanied by an increase in the oxidative capacity in Thoroughbreds.