Impact of 5 fluorouracil chemotherapy on gut inflammation, functional parameters, and gut microbiota.

Emerging evidence suggests that gut microbiota may influence the response to chemotherapy. We sought to characterize the effects of 5 fluorouracil (5FU) chemotherapy on colon inflammation and functional measures in colorectal cancer (CRC) and to further determine whether gut microbiota can influence this response. 50 C57BL/6 were randomized into four groups; Control + Vehicle (n = 10), Control + 5FU (n = 10), AOM/DSS + Vehicle (n = 15), and AOM/DSS + 5FU (n = 15). CRC was induced chemically by a single 10 mg/kg injection of azoxymethane (AOM) followed by two cycles (2% and 1%) of dextran sodium sulfate (DSS). Mice were then treated with 3 cycles of vehicle or 5FU (cycle 1: 40 mg/kg, cycle 2 + 3: 20 mg/kg). Functional tests (grip strength and run-to-fatigue) were performed prior to 5FU treatment (baseline) and at the completion of the second cycle of 5FU. Following the third 5FU cycle, mice were euthanized and the colon was evaluated for expression of inflammatory genes using RT-qPCR and stool samples were profiled using 16S rRNA sequencing. A second experiment used fecal microbiota transplantation from 5FU treated mice to control mice (n = 10-15/group) to determine whether 5FU associated changes in the microbiota could influence functional measures and colon inflammation. 5FU reduced grip strength (p < 0.05) and caused a trending decrease in run-to-fatigue performance in cancer mice (p = 0.06). Select intestinal inflammatory genes were significantly elevated with 5FU treatment and this was further exacerbated with cancer (p < 0.05). Microbiota analysis revealed increased dissimilarity and alterations in bacterial taxonomy in 5FU and AOM/DSS-treated mice (p < 0.05). Fecal transplant from 5FU treated mice reduced functional performance (p < 0.05) and altered select colon inflammatory markers (p < 0.05). This study provides evidence of an effect of 5FU on inflammatory responses and functional measures in a mouse model of CRC and suggests that gut microbes may play a role in some, but not all, 5FU related perturbations.

PGC-1α4 gene expression is suppressed by the IL-6-MEK-ERK 1/2 MAPK signalling axis and altered by resistance exercise, obesity and muscle injury.

AIM: PGC-1α4 is a novel regulator of muscle hypertrophy; however, there is limited understanding of the regulation of its expression and role in many (patho)physiological conditions. Therefore, our purpose was to elicit signalling mechanisms regulating gene expression of Pgc1α4 and examine its response to (patho)physiological stimuli associated with altered muscle mass. METHODS: IL-6 knockout mice and pharmacological experiments in C2C12 myocytes were used to identify regulation of Pgc1α4 transcription. To examine Pgc1α4 gene expression in (patho)physiological conditions, obese and lean Zucker rats with/without resistance exercise (RE), ageing mice and muscle regeneration from injury were examined. RESULTS: In IL-6 knockout mice, Pgc1α4mRNA was ~sevenfold greater than wild type. In C2C12 cells, Pgc1α4mRNA was suppressed ~70% by IL-6. Suppression of Pgc1α4 by IL-6 was prevented by MEK-ERK-MAPK inhibition. RE led to ~260% greater Pgc1α4mRNA content in lean rats. However, obese Zucker rats exhibited ~270% greater Pgc1α4mRNA than lean, sedentary with no further augmentation by RE. No difference was seen in IL-6mRNA or ERK-MAPK phosphorylation in Zucker rats. Aged mice demonstrated ~50% lower Pgc1α4mRNA and ~fivefold greater ERK-MAPK phosphorylation than young despite unchanged Il-6mRNA. During muscle regeneration, Pgc1α4 content is ~30% and IL-6mRNA >threefold of uninjured controls 3 days following injury; at 5 days, Pgc1α4 was >twofold greater in injured mice with no difference in IL-6mRNA. CONCLUSION: Our findings reveal a novel mechanism suppressing Pgc1α4 gene expression via IL-6-ERK-MAPK and suggest this signalling axis may inhibit Pgc1α4 in some, but not all, (patho)physiological conditions.

Altered cardiac muscle mTOR regulation during the progression of cancer cachexia in the ApcMin/+ mouse.

Cancer cachexia is a muscle wasting condition that occurs in response to a malignant growth in the body. The mechanisms regulating cardiac muscle mass with cachexia are not well understood. Using the ApcMin/+ mouse model of colorectal cancer, we investigated how cachexia affects the regulation of 5'-adenosine monophosphate-activated protein kinase (AMPK), protein kinase B (Akt) and mammalian target of rapamycin (mTOR) signaling in the heart. Compared to age-matched C57BL/6 (BL6) mice, ApcMin/+ body mass and heart mass were lower at 12 (11 ± 5 and 8 ± 3%, respectively) and 20 weeks (26 ± 3 and 6 ± 4%, respectively) of age (P<0.05). Diminished heart mass in the 20-week-old ApcMin/+ mice coincided with a decreased rate of myofibrillar protein synthesis and increased AMPKα phosphorylation. Cachexia decreased mTOR phosphorylation and the phosphorylation of the mTOR substrates, S6 ribosomal protein and 4EBP1 independent of Akt activation. These changes in mTOR-related protein signaling were accompanied by modest increases in the amount of Beclin1 but not protein ubiquitination or cardiomyocyte apoptosis. Taken together, these data suggest that loss of cardiac mass during cachexia progression in the ApcMin/+ mouse is associated with an Akt-independent suppression of anabolic signaling and evidence of increased autophagy.

Skeletal muscle mass recovery from atrophy in IL-6 knockout mice.

AIM: Skeletal muscle interleukin-6 (IL-6) expression is induced by continuous contraction, overload-induced hypertrophy and during muscle regeneration. The loss of IL-6 can alter skeletal muscle's growth and extracellular matrix remodelling response to overload-induced hypertrophy. Insulin-like growth factor-1 (IGF-1) gene expression and related signalling through Akt/mTOR is a critical regulator of muscle mass. The significance of IL-6 expression during the recovery from muscle atrophy is unclear. This study's purpose was to determine the effect of IL-6 loss on mouse gastrocnemius (GAS) muscle mass during recovery from hindlimb suspension (HS)-induced atrophy. METHODS: Female C57BL/6 [wild type (WT)] and IL-6 knockout (IL-6 KO) mice at 10 weeks of age were assigned to control, HS or HS followed by normal cage ambulation groups. RESULTS: GAS muscle atrophy was induced by 10 days of HS. HS induced a 20% loss of GAS mass in both WT and IL-6 KO mice. HS+7 days of recovery restored WT GAS mass to cage-control values. GAS mass from IL-6 KO mice did not return to cage-control values until HS+14 days of recovery. Both IGF-1 mRNA expression and Akt/mTOR signalling were increased in WT muscle after 1 day of recovery. In IL-6 KO muscle, IGF-1 mRNA expression was decreased and Akt/mTOR signalling was not induced after 1 day of recovery. MyoD and myogenin mRNA expression were both induced in WT muscle after 1 day of recovery, but not in IL-6 KO muscle. CONCLUSION: Muscle IL-6 expression appears important for the initial growth response during the recovery from disuse.

Overload-induced skeletal muscle extracellular matrix remodelling and myofibre growth in mice lacking IL-6.

AIM: Overloading healthy skeletal muscle produces myofibre hypertrophy and extracellular matrix remodelling, and these processes are thought to be interdependent for producing muscle growth. Inflammatory cytokine interleukin-6 (IL-6) gene expression is induced in overloaded skeletal muscle, and the loss of this IL-6 induction can attenuate the hypertrophic response to overload (OV). Although the OV induction of IL-6 in skeletal muscle may be an important regulator of inflammatory processes and satellite cell proliferation, less is known about its role in the regulation of extracellular matrix remodelling. The purpose of the current study was to examine if OV-induced extracellular matrix remodelling, muscle growth, and associated gene expression were altered in mice that lack IL-6, when compared with wild-type mice. METHODS: Male C57/BL6 (WT) and C57/BL6 x IL-6(-/-) (IL-6(-/-)) mice (10 weeks of age) were assigned to either a sham control or synergist ablation OV treatments for 3, 21 or 56 days. RESULT: Plantaris muscle mass increased 59% in WT and 116% in IL-6(-/-) mice after 21 day OV. Myofibre CSA was also increased by 21 day OV in both WT and IL-6(-/-) mice. OV induced a twofold greater increase in the volume of non-contractile tissue in IL-6(-/-) muscle compared to WT. OV also induced a significantly greater accumulation of hydroxyproline and procollagen-1 mRNA in IL-6(-/-) muscle, when compared with WT muscle after 21 day OV. Transforming growth factor-beta and insulin-like growth factor-1 mRNA expression were also induced to a greater extent in IL-6(-/-) muscle when compared with WT muscle after 21 day OV. There was no effect of IL-6 loss on the induction of myogenin, and cyclin D1 mRNA expression after 3 day OV. However, MyoD mRNA expression in 3 day OV IL-6(-/-) muscle was attenuated when compared with WT OV mice. CONCLUSION: IL-6 appears to be necessary for the normal regulation of extracellular matrix remodelling during OV-induced growth.

Benefits of oat beta-glucan on respiratory infection following exercise stress: role of lung macrophages.

Exercise stress is associated with an increased risk for upper respiratory tract infection (URTI). We have shown that consumption of the soluble oat fiber beta-glucan (ObetaG) can offset the increased risk for infection and decreased macrophage antiviral resistance following stressful exercise; however, the direct role of macrophages is unknown. This study examined the effect of macrophage depletion on the benefits of orally administered ObetaG on susceptibility to infection (morbidity, symptom severity, and mortality) following exercise stress. CL(2)MDP (Ex- H(2)O-CL(2)MDP, Ex-ObetaG-CL(2)MDP, Con-H(2)O-CL(2)MDP, Con-ObetaG-CL(2)MDP)-encapsulated liposomes were administered intranasally to deplete macrophages, and PBS (Ex-H(2)O-PBS, Ex-ObetaG-PBS, Con-H(2)O-PBS, Con-ObetaG-PBS)-encapsulated liposomes were given to macrophage-intact groups. Ex mice ran to volitional fatigue on a treadmill for 3 consecutive days, and ObetaG mice were fed a solution of 50% ObetaG in their drinking water for 10 consecutive days before infection. Fifteen minutes following the final bout of Ex or rest, mice were intranasally inoculated with 50 microl of a standardized dose of herpes simplex virus-1. Ex increased morbidity (P < 0.001) and symptom severity (P < 0.05) but not mortality (P = 0.09). The increase in morbidity and symptom severity was blocked by ObetaG consumption for 10 consecutive days before exercise and infection [morbidity (P < 0.001) and symptom severity (P < 0.05)]. Depletion of macrophages negated the beneficial effects of ObetaG on reducing susceptibility to infection following exercise stress, as evidenced by an increase in morbidity (P < 0.01) and symptom severity (P < 0.05). Results indicate that lung macrophages are at least partially responsible for mediating the beneficial effects of ObetaG on susceptibility to respiratory infection following exercise stress.

Susceptibility to HSV-1 infection and exercise stress in female mice: role of estrogen.

Exhaustive exercise has been associated with an increased risk for upper respiratory tract infections in mice and humans. We have previously shown (Brown AS, Davis JM, Murphy AE, Carmichael MD, Ghaffer A, Mayer EP. Med Sci Sports Exerc 36: 1290-1295, 2004) that female mice are better protected from the lethal effects of herpes simplex virus type 1 (HSV-1) infection, both at rest and following exercise stress, but little is known about possible mechanisms. This study tested the effects of estrogen on HSV-1 infection and macrophage antiviral resistance following repeated exhaustive exercise. Female mice were assigned to either exercise (Ex) or control (C): intact female (I-C or I-Ex), ovariectomized female (O-C or O-Ex), or ovariectomized estrogen-supplemented female (E-C or E-Ex). Exercise consisted of treadmill running to volitional fatigue ( approximately 125 min) for 3 consecutive days. Intact female mice had a later time to death than O and E (P < 0.05) and fewer deaths than both O and E (P < 0.05). Exercise stress was associated with increased time to sickness (P < 0.05) and symptom severity at days 6 and 12-21 postinfection (P < 0.05) and decreased macrophage antiviral resistance (P < 0.001) in all groups. E had increased symptom severity at days 6 and 13-21 postinfection (P < 0.05). Results indicate that intact female mice are better protected from the lethal effects of HSV-1 infection and that exercise stress had a similar negative impact in all groups. This protective effect was lost in ovariectomized mice, but it was not reinstated by 17beta-estradiol replacement. This indicates that other ovarian factors, alone or in combination with estrogen, are responsible for the protective effects in females.

Ovarian hormone status and skeletal muscle inflammation during recovery from disuse in rats.

Resumption of normal muscle loading after a period of disuse initiates cellular processes related to mass accretion. The renewed loading also induces a significant amount of muscle damage and subsequent inflammation. Ovarian hormone depletion delays atrophied myofibre mass recovery. Ovarian hormones are also global regulators of immune system function. The purpose of this study was to determine whether ovarian hormone depletion-induced deficits in myofibre regrowth after disuse atrophy are related to the induction of muscle damage and the associated inflammatory response. We hypothesized that soleus muscle immune cell infiltration and inflammatory gene expression would be both accentuated and prolonged in ovarian hormone-depleted rats during the first week of recovery from disuse atrophy. Intact and ovariectomized (OVX) female rats were subjected to hindlimb suspension for 10 days and then returned to normal ambulation for a recovery period, the rats were killed and the soleus muscle removed for analysis. Although reloading increased both circulating creatine kinase and myofibre membrane disruption, there was no effect of ovarian hormones on these processes during recovery. Muscle neutrophil concentration was increased above baseline regardless of hormone status at days 1 and 3 of recovery; however, this increase was 43% greater at day 3 in the OVX group. Muscle ED1+ and ED2+ macrophage concentrations were increased during recovery in both groups. However, macropage concentrations remained elevated at day 7 of recovery in the OVX group, whereas they returned to control levels in the intact group. Cyclo-oxygenase-2, interleukin-6 and interleukin-1beta muscle mRNA expression increased similarly during recovery, regardless of ovarian hormone status. These results demonstrate that the initial myofibre damage and inflammatory gene expression induced during muscle recovery from disuse atrophy are independent of ovarian hormone status.

Expression of an Nkx3.1-CRE gene using ROSA26 reporter mice.

The genetic locus of Nkx3.1, an early murine marker of sclerotome and prostate development, was disrupted by a knock in of CRE recombinase via homologous recombination in embryonic stem cells. Cell fate mapping revealed previously unidentified cell lineages expanded from Nkx3.1-expressing cell populations and recapitulated reported Nkx3.1 expression patterns. In lineage trace experiments of E18.5 Nkx3.1-CRE; R26R embryos novel staining was observed in areas of the lungs, portions of the duodenum, and vertebral elements of the skeleton. beta-galactosidase activity measured in Nkx3.1-CRE; R26R and Nkx3.2-CRE; R26R embryos was observed in overlapping regions of the sclerotome but no apparent change in Nkx3.1 expression was seen in the Nkx3.2 mutants by in situ hybridization.

Counteracting muscle wasting in HIV-infected individuals.

HIV-infected persons often experience a loss of lean tissue mass, which includes decreases in skeletal muscle mass. This HIV-associated wasting is significant because it has been associated with accelerated disease progression and increased morbidity. Signalling related to several circulating molecules, including tumour necrosis factor (TNF)-alpha, growth hormone, insulin-like growth factor (IGF)-1 and testosterone, has been associated with the aetiology of muscle wasting. Additionally, nutritional status related to malnutrition and specific dietary deficiencies may be involved. In an attempt to counter muscle wasting in HIV-infected persons, treatments have been suggested that target these mechanisms. Nutritional supplementation, cytokine reduction, hormone therapy and resistance exercise training are potential treatments for this condition. Resistance exercise training, which is more easily accessible to this population than other treatments, holds promise in counteracting the process of HIV wasting, as it has been successfully used to increase lean tissue mass in healthy and clinical populations. This review will explore the HIV/AIDS muscle-wasting syndrome, its aetiology, and the treatments used to counteract wasting.

Estrogen status and skeletal muscle recovery from disuse atrophy.

Although estrogen loss can alter skeletal muscle recovery from disuse, the specific components of muscle regrowth that are estrogen sensitive have not been described. The primary purpose of this study was to determine the components of skeletal muscle mass recovery that are biological targets of estrogen. Intact, ovariectomized (OVX), and ovariectomized with 17beta-estradiol replacement (OVX+E2) female rats were subjected to hindlimb suspension for 10 days and then returned to normal cage ambulation for the duration of recovery. Soleus muscle mass returned to control levels by day 7 of recovery in the intact animals, whereas OVX soleus mass did not recover until day 14. Intact rats recovered soleus mean myofiber cross-sectional area (CSA) by day 14 of recovery, whereas the OVX soleus remained decreased (42%) at day 14. OVX mean fiber CSA did return to control levels by day 28 of recovery. The OVX+E2 treatment group recovered mean CSA at day 14, as in the intact animals. Myofibers demonstrating central nuclei were increased at day 14 in the OVX group, but not in intact or OVX+E2 animals. The percent noncontractile tissue was also increased 29% in OVX muscle at day 14, but not in either intact or OVX+E2 groups. In addition, collagen 1a mRNA was increased 45% in OVX muscle at day 14 of recovery. These results suggest that myofiber growth, myofiber regeneration, and extracellular matrix remodeling are estrogen-sensitive components of soleus muscle mass recovery from disuse atrophy.

RhoA expression during recovery from skeletal muscle disuse.

Functional overload and anabolic steroid administration induce signaling pathways that regulate skeletal muscle RhoA expression. The purpose of this study was to determine RhoA and associated protein expression at the onset of disuse and after a brief period of reloading. Male Sprague-Dawley rats were randomly assigned to cage control (Con), 3 days of hindlimb suspension (Sus), or 3 days of hindlimb suspension with 12 h of reloading (12-h Reload). The reloading stimuli consisted of 12 h of resumed normal locomotion after 3 days of hindlimb suspension. Plantaris muscle-to-body weight (mg/g) ratio decreased 17% from Con with Sus but returned to Con with 12-h Reload, increasing 13% from Sus. Sus decreased RhoA protein concentration 46%, whereas 12-h Reload induced a 24% increase compared with Sus. The ratio of cytosolic- to membrane-associated RhoA protein was not changed with either Sus or 12-h Reload. RhoA mRNA concentration was decreased 48% by Sus, and 12-h Reload induced a 170% increase from Sus. beta(1)-Integrin protein, a transmembrane protein associated with RhoA activation, was not altered by Sus but increased 155% with 12-h Reload. Although beta(1)-integrin mRNA was not altered by Sus, it increased 70% from Con with 12-h Reload. Rho family member Cdc42 protein associated with the muscle membrane was decreased 60% with Sus, and 12-h Reload induced a 172% increase compared with Sus. In conclusion, decreased RhoA protein expression and mRNA abundance are early adaptations to disuse but recover rapidly after normal locomotion is resumed.

Effect of creatine supplementation on cardiac muscle of exercise-stressed rats.

The role of creatine supplementation in altering the physiological parameters regulating cardiac muscle's functional capacity through the initiation of cardiac hypertrophy and altered contractile protein expression has not been determined. The purpose of this study was to determine the effect of creatine supplementation, with and without exercise stress, on physiological parameters regulating functional capacity through alterations in rat cardiac mass and contractile-protein expression. Thirty male Sprague-Dawley rats were subjected to 30 min of exercise stress 5 days/week for 3 weeks with 2% of total body mass attached to the tail. Animals were randomly assigned to one of four treatment groups: group 1 (Con) received (1 ml/day) sucrose water by intubation tube (n=8); group 2 (Cr) received (1 ml/day) sucrose/creatine solution (n=6); group 3 (EX) received 1 ml/day sucrose water and the exercise stimulus (n=8), and group 4 (Cr/EX) received (1 ml/day) sucrose/creatine solution and the exercise stimulus (n=8). At the conclusion of the 21-day exercise-training period, the heart was collected and weighed for determination of wet weight, total protein, total RNA, and myosin heavy chain protein expression. RNA concentration decreased significantly (13%) in the EX group, but not in the CR/EX group, indicating an interactive effect of creatine and exercise. Total RNA significantly decreased (15%) in the EX group. Protein concentration significantly increased (9%) in the exercising treatments, while total protein did not change. Cardiac myosin heavy chain expression significantly shifted towards a predominant expression of the beta-isoform in the Cr/EX group [54.53% (3.42) beta]. These results indicate an interaction of creatine supplementation and swimming exercise stress that potentially alters cardiac protein synthesis and demonstrates a possible mechanism through which the combination of creatine supplementation and swimming stress stimuli act to alter the physiological parameters regulating cardiac functional capacity.

Carbohydrate ingestion influences skeletal muscle cytokine mRNA and plasma cytokine levels after a 3-h run.

Sixteen experienced marathoners ran on treadmills for 3 h at approximately 70% maximal oxygen consumption (Vo(2 max)) on two occasions while receiving 1 l/h carbohydrate (CHO) or placebo (Pla) beverages. Blood and vastus lateralis muscle biopsy samples were collected before and after exercise. Plasma was analyzed for IL-6, IL-10, IL-1 receptor agonist (IL-1ra), IL-8, cortisol, glucose, and insulin. Muscle was analyzed for glycogen content and relative gene expression of 13 cytokines by using real-time quantitative RT-PCR. Plasma glucose and insulin were higher, and cortisol, IL-6, IL-10, and IL-1ra, but not IL-8, were significantly lower postexercise in CHO vs. Pla. Change in muscle glycogen content did not differ between CHO and Pla (P = 0.246). Muscle cytokine mRNA content was detected preexercise for seven cytokines in this order (highest to lowest): IL-15, TNF-alpha, IL-8, IL-1beta, IL-12p35, IL-6, and IFN-gamma. After subjects ran for 3 h, gene expression above prerun levels was measured for five of these cytokines: IL-1beta, IL-6, and IL-8 (large increases), and IL-10 and TNF-alpha (small increases). The increase in mRNA (fold difference from preexercise) was attenuated in CHO (15.9-fold) compared with Pla (35.2-fold) for IL-6 (P = 0.071) and IL-8 (CHO, 7.8-fold; Pla, 23.3-fold; P = 0.063). CHO compared with Pla beverage ingestion attenuates the increase in plasma IL-6, IL-10, and IL-1ra and gene expression for IL-6 and IL-8 in athletes running 3 h at 70% Vo(2 max) despite no differences in muscle glycogen content.

beta1 integrin and organized actin filaments facilitate cardiomyocyte-specific RhoA-dependent activation of the skeletal alpha-actin promoter.

Activation of RhoA GTPase causes actin filament bundling into stress fibers, integrin clustering, and focal adhesion formation through its action on actin cytoskeleton organization. RhoA also regulates transcriptional activity of serum response factor (SRF). Recent studies in NIH 3T3 fibroblasts have shown that SRF activation by RhoA does not require an organized cytoskeleton and may be regulated by G-actin level. In cardiac myocytes, the organization of actin fibers into myofibrils is one of the primary characteristics of cardiac differentiation and hypertrophy. The primary purpose of this study was to examine if RhoA regulates SRF-dependent gene expression in neonatal cardiomyocytes in a manner different from that observed in fibroblasts. Our results show that RhoA-dependent skeletal alpha-actin promoter activation requires beta1 integrin and a functional cytoskeleton in cardiomyocytes but not in NIH 3T3 fibroblasts. Activation of the alpha-actin promoter by RhoA is greatly potentiated (up to 15-fold) by co-expression of the integrin beta1A or beta1D isoform but is significantly reduced by 70% with a co-expressed dominant negative mutant of beta1 integrin. Furthermore, clustering of beta1 integrin with anti-beta1 integrin antibodies potentiates synergistic RhoA and beta1 integrin activation of the alpha-actin promoter. Cytochalasin D and latrunculin B, inhibitors of actin polymerization, significantly reduced RhoA-induced activation of the alpha-actin promoter. Jasplakinolide, an actin polymerizing agent, mimics the synergistic effect of RhoA and beta1 integrin on the actin promoter. These observations support the concept that RhoA regulates SRF-dependent cardiac gene expression through cross-talk with beta1 integrin signal pathway via an organized actin cytoskeleton.

The smooth muscle gamma-actin gene promoter is a molecular target for the mouse bagpipe homologue, mNkx3-1, and serum response factor.

An evolutionarily conserved vertebrate homologue of the Drosophila NK-3 homeodomain gene bagpipe, Nkx3-1, is expressed in vascular and visceral mesoderm-derived muscle tissues and may influence smooth muscle cell differentiation. Nkx3-1 was evaluated for mediating smooth muscle gamma-actin (SMGA) gene activity, a specific marker of smooth muscle differentiation. Expression of mNkx3-1 in heterologous CV-1 fibroblasts was unable to elicit SMGA promoter activity but required the coexpression of serum response factor (SRF) to activate robust SMGA transcription. A novel complex element containing a juxtaposed Nkx-binding site (NKE) and an SRF-binding element (SRE) in the proximal promoter region was found to be necessary for the Nkx3-1/SRF coactivation of SMGA transcription. Furthermore, Nkx3-1 and SRF associate through protein-protein interactions and the homeodomain region of Nkx3-1 facilitated SRF binding to the complex NKE.SRE. Mutagenesis of Nkx3-1 revealed an inhibitory domain within its C-terminal segment. In addition, mNkx3-1/SRF cooperative activity required an intact Nkx3-1 homeodomain along with the MADS box of SRF, which contains DNA binding and dimerization structural domains, and the contiguous C-terminal SRF activation domain. Thus, SMGA is a novel target for Nkx3-1, and the activity of Nkx3-1 on the SMGA promoter is dependent upon SRF.

The neprilysin family in health and disease.

The mammalian neprilysin (NEP) family comprises at least seven members: NEP itself, Kell blood group antigen (KELL), the endothelin-converting enzymes (ECE-1 and ECE-2), the enzyme PEX, associated with X-linked hypophosphataemia, "X-converting enzyme" (XCE) a CNS-expressed orphan peptidase and a soluble, secreted endopeptidase (SEP). These zinc metallopeptidases are all type II integral membrane proteins. Where identified, these enzymes have roles in the processing or metabolism of regulatory peptides and therefore represent potential therapeutic targets. A distinct feature of ECE-1 species is their existence as distinct isoforms differing in their N-terminal cytoplasmic tails. These tails play a role in enzyme targeting and turnover with di-leucine and tyrosine-based motifs affecting localization. Additional anchorage of these enzymes can also occur through palmitoylation. Bacterial homologues of the neprilysin family exist, for example the products of the pepO genes from L. lactis and S. parasanguis, and a recently described gene product of P. gingivalis which is an ECE-1 homologue that can catalyse the conversion of big endothelin to endothelin. A genomics based approach to understanding the functions of this proteinase family is aided by the completion of the C. elegans and Drosophila genomes, both of which encode multiple copies of NEP-like enzymes.

Integrin signaling's potential for mediating gene expression in hypertrophying skeletal muscle.

Overloaded skeletal muscle undergoes dramatic shifts in gene expression, which alter both the phenotype and mass. Molecular biology techniques employing both in vivo and in vitro hypertrophy models have demonstrated that mechanical forces can alter skeletal muscle gene regulation. This review's purpose is to support integrin-mediated signaling as a candidate for mechanical load-induced hypertrophy. Research quantifying components of the integrin-signaling pathway in overloaded skeletal muscle have been integrated with knowledge regarding integrins role during development and cardiac hypertrophy, with the hope of demonstrating the pathway's importance. The role of integrin signaling as an integrator of mechanical forces and growth factor signaling during hypertrophy is discussed. Specific components of integrin signaling, including focal adhesion kinase and low-molecular-weight GTPase Rho are mentioned as downstream targets of this signaling pathway. There is a need for additional mechanistic studies capable of providing a stronger linkage between integrin-mediated signaling and skeletal muscle hypertrophy; however, there appears to be abundant justification for this type of research.

Focal adhesion proteins FAK and paxillin increase in hypertrophied skeletal muscle.

Components of signaling pathways for mechanotransduction during load-induced enlargement of skeletal muscle have not been completely defined. We hypothesized that loading of skeletal muscle would result in an adaptive increase in the expression of two focal adhesion complex (FAC)-related proteins, focal adhesion kinase (FAK) and paxillin, as well as increased FAK activity. FAK protein was immunolocalized to the sarcolemmal region of rooster anterior latissimus dorsi (ALD) myofibers in the middle of the ALD muscle. FAK (77 and 81%) and paxillin (206 and 202%) protein concentrations per unit of total protein in Western blots increased significantly after 1.5 and 7 days, but not after 13 days, of stretch-induced hypertrophy-hyperplasia of the ALD muscle. FAK autokinase activity in immunoprecipitates was increased after 1.5, 7, and 13 days in stretched ALD muscles. To determine whether increased FAK and paxillin protein concentrations are associated with hypertrophy and/or new fiber formation, two additional experiments were performed. First, during formation of primary chicken myotubes (a model of new fiber formation), FAK protein concentration (63%), FAK activity (157%), and paxillin protein concentration (97%) increased compared with myoblasts. Second, FAK (112% and 611%) and paxillin (87% and 431%) protein concentrations per unit of total protein in the soleus muscle increased at 1 and 8 days after surgical ablation of the synergistic gastrocnemius muscle (a model of hypertrophy without hyperplasia). Thus increases in components of the FAC occur in hypertrophying muscle of animals and in newly formed muscle fibers in culture. Furthermore, increased FAK activity suggests a possible convergence of signaling at the FAC in load-induced growth of skeletal muscle.