Function of mitochondrial Stat3 in cellular respiration.

Cytokines such as interleukin-6 induce tyrosine and serine phosphorylation of Stat3 that results in activation of Stat3-responsive genes. We provide evidence that Stat3 is present in the mitochondria of cultured cells and primary tissues, including the liver and heart. In Stat3(-/-) cells, the activities of complexes I and II of the electron transport chain (ETC) were significantly decreased. We identified Stat3 mutants that selectively restored the protein's function as a transcription factor or its functions within the ETC. In mice that do not express Stat3 in the heart, there were also selective defects in the activities of complexes I and II of the ETC. These data indicate that Stat3 is required for optimal function of the ETC, which may allow it to orchestrate responses to cellular homeostasis.

PKCalpha regulates phosphorylation and enzymatic activity of cPLA2 in vitro and in activated human monocytes.

Phospholipases A(2) (PLA(2)) are potent regulators of the inflammatory response. We have observed that Group IV cPLA(2) activity is required for the production of superoxide anion (O(2)(-)) in human monocytes [Li Q., Cathcart M.K. J. Biol. Chem. 272 (4) (1997) 2404-2411.]. We have previously identified PKCalpha as a kinase pathway required for monocyte O(2)(-) production [Li Q., Cathcart M.K. J. Biol. Chem. 269 (26) (1994) 17508-17515.]. We therefore investigated the potential interaction between PKCalpha and cPLA(2) by evaluating the requirement for specific PKC isoenzymes in the process of activating cPLA(2) enzymatic activity and protein phosphorylation upon monocyte activation. We first showed that general PKC inhibitors and antisense oligodeoxyribonucleotides (ODN) to the cPKC group of PKC enzymes inhibited cPLA(2) activity. To distinguish between PKCalpha and PKCbeta isoenzymes in regulating cPLA(2) protein phosphorylation and enzymatic activity, we employed our previously characterized PKCalpha or PKCbeta isoenzyme-specific antisense ODN [Li Q., Subbulakshmi V., Fields A.P., Murray, N.R., Cathcart M.K., J. Biol. Chem. 274 (6) (1999) 3764-3771]. Suppression of PKCalpha expression, but not PKCbeta expression, inhibited cPLA(2) protein phosphorylation and enzymatic activity. Additional studies ruled out a contribution by Erk1/2 to cPLA(2) phosphorylation and activation. We also found that cPLA(2) co-immunoprecipitated with PKCalpha and vice versa. In vitro studies demonstrated that PKCalpha could directly phosphorylate cPLA(2).and enhance enzymatic activity. Finally, we showed that addition of arachidonic acid restored the production of O(2)(-) in monocytes defective in either PKCalpha or cPLA(2) expression. Taken together, our data suggest that PKCalpha, but not PKCbeta, is the predominant cPKC isoenzyme required for cPLA(2) protein phosphorylation and maximal induction of cPLA(2) enzymatic activity upon activation of human monocytes. Our data also support the concept that the requirements for PKCalpha and cPLA(2) in O(2)(-) generation are solely due to their seminal role in generating arachidonic acid.

Structural and functional consequences of c-N-Ras constitutively associated with intact mitochondria.

We demonstrate that both c-N-Ras and c-K(B)-Ras are constitutively associated with purified mitochondria. c-K(B)-Ras is associated with the mitochondrial outer membrane, and c-N-Ras is associated with both the outer membrane and inner mitochondrial compartments. The mitochondrial morphology is abnormal in both c-N-Ras negative and K-Ras negative cells. Normal mitochondrial morphology was restored by targeting N-Ras to both the inner and outer mitochondrial compartments, or by ectopically expressing c-K(B)-Ras. Impaired mitochondrial function can result in increased CHOP and NFkappaB activity, typical for a retrograde signaling response. Both are constitutively elevated in the N-Ras negative cells, but not in the K-Ras negative background, and are restored by c-N-Ras targeted exclusively to the inner mitochondrial compartment. Surprisingly, both targeting and the ability to functionally reduce retrograde transcriptional activity were found to be independent of c-N-Ras farnesylation. Overall, these data demonstrate for the first time a (1) farnesylation independent function for c-N-Ras and (2) that N-Ras within the inner mitochondrial compartment is an essential component of the retrograde signaling system between the mitochondria and nucleus.

Tyk2 tyrosine kinase expression is required for the maintenance of mitochondrial respiration in primary pro-B lymphocytes.

Tyk2, a member of the Jak family of protein tyrosine kinases, is critical for the biological actions of alpha/beta interferon (IFN-alpha/beta). Although Tyk2(-/-) mice are phenotypically normal, they exhibit abnormal responses to inflammatory challenges in a variety of cells isolated from Tyk2(-/-) mice. The reported phenotypic alterations in both Tyk2-null cells and mice are consistent with the possibility that the expression of this tyrosine kinase may regulate mitochondrial function. We report here that Tyk2-null pro-B cells are markedly deficient in basal oxygen consumption and exhibit a significant decrease in steady-state cellular ATP levels compared to wild-type cells. Tyk2-null cells also exhibit impaired complex I, III, and IV function of the mitochondrial electron transport chain. Reconstitution of Tyk2-null pro-B cells with either the wild type or a kinase-inactive mutant of Tyk2 restores basal mitochondrial respiration. By contrast, the kinase activity of Tyk2 is required for maintenance of both complex I-dependent mitochondrial respiration as well as induction of apoptosis in cells incubated with IFN-beta. Consistent with the role of Tyk2 in the regulation of tyrosine phosphorylation of Stat3, expression of a constitutively active Stat3 can restore the mitochondrial respiration in Tyk2-null cells treated with IFN-beta. Finally, Tyk2(-/-) mice show decreased exercise tolerance compared to wild-type littermates. Our results implicate a novel role for Tyk2 kinase and Stat3 phosphorylation in mitochondrial respiration.

Growth factor-dependent AKT activation and cell migration requires the function of c-K(B)-Ras versus other cellular ras isoforms.

K-Ras-negative fibroblasts are defective in their steady-state expression of MMP-2. This occurs through c-K(B)-Ras dependent regulation of basal levels of AKT activity. In this report, we have extended those studies to demonstrate that in the absence of K-Ras expression, PDGF-BB fails to induce significant AKT activation, although this was not the case in N-Ras-negative cells. This phenotype was directly linked to PDGF-dependent cell migration. All of the independently immortalized K-Ras-negative cells failed to migrate upon the addition of PDGF. Only ectopic expression of c-K(B)-Ras, not c-K(A)-Ras nor oncogenic N-Ras, could restore both PDGF-dependent AKT activation and cell migration. Since most Ras binding partners can interact with all Ras isoforms, the specificity of PDGF-dependent activation of AKT and enhanced cell migration suggests that these outcomes are likely to be regulated through a c-K(B)-Ras-specific binding partner. Others have published that of the four Ras isoforms, only K(B)-Ras can form a stable complex with calmodulin (CaM). Along those lines, we provide evidence that 1) PDGF addition results in increased levels of a complex between c-K(B)-Ras and CaM and 2) the biological outcomes that are strictly dependent on c-K(B)-Ras (AKT activation and cell migration) are blocked by CaM antagonists. The PDGF-dependent activation of ERK is unaffected by the absence of K(B)-Ras and presence of CaM antagonists. This is the first example of a linkage between a specific biological outcome, cell migration, and the activity of a single Ras isoform, c-K(B)-Ras.

Decibel attenuation of pulsed electromagnetic field (PEMF) in blood and cortical bone determined experimentally and from the theory of ohmic losses.

We studied the PEMF power attenuation in tissues representative of clinical applications (blood and cortical bone) to determine the amount of power available for PEMF purported biological effects. The experimental system consisted of a pair of nearly circular, parallel and coaxial coils separated by a distance of one coil diameter. The power attenuation was measured using a small search coil connected to a digital oscilloscope. The coils were powered by a voltage switch operating at two different frequencies (3.8 and 63 kHz) producing bursts of pulses (numbering 21 and 1619) and triggered at two different frequencies (1.5 and 15 Hz, respectively). The tissue samples were placed inside the coils so as to expose them to either transverse electric field (at the center of coils) or the transverse magnetic field (at the coil wire). The cylindrical coil geometry yielded closed-form expressions for power attenuation based on magnetic diffusion equation and ohmic losses due to bulk tissue magnetic permeability and electrical conductivity. The measured power attenuation at these PEMF frequencies of not more than one decibel (1 dB) was well explained by the theory for the 3.8 kHz but less so for the 63 kHz frequency PEMF. The results provide important insights regarding physical mechanism of weak PEMF power dissipation in tissues.

Exposure of murine cells to pulsed electromagnetic fields rapidly activates the mTOR signaling pathway.

Murine pre-osteoblasts and fibroblast cell lines were used to determine the effect of pulsed electromagnetic field (PEMF) exposure on the production of autocrine growth factors and the activation of early signal transduction pathways. Exposure of pre-osteoblast cells to PEMF minimally increased the amount of secreted TGF-beta after 1 day, but had no significant effects thereafter. PEMF exposure of pre-osteoblast cells also had no effect on the amount of prostaglandin E(2) in the conditioned medium. Exposure of both pre-osteoblasts and fibroblasts to PEMF rapidly activated the mTOR signaling pathway, as evidenced by increased phosphorylation of mTOR, p70 S6 kinase, and the ribosomal protein S6. Inhibition of PI3-kinase activity with the chemical inhibitor LY294002 blocked PEMF-dependent activation of mTOR in both the pre-osteoblast and fibroblast cell lines. These findings suggest that PEMF exposure might function in a manner analogous to soluble growth factors by activating a unique set of signaling pathways, inclusive of the PI-3 kinase/mTOR pathway.

Exposure of mouse preosteoblasts to pulsed electromagnetic fields reduces the amount of mature, type I collagen in the extracellular matrix.

We tested the hypothesis that exposure of a mouse preosteoblast cell line to pulsed electromagnetic fields (PEMF) would affect components of the extracellular matrix. We report that exposure of MC3T3-E1 cells to a single PEMF waveform significantly reduced the amount of mature, alpha1(I) collagen in the extracellular matrix (ECM) and the conditioned medium, without affecting the amount of total ECM protein. This decrease was not due to changes in the steady-state level of Col1A1 mRNA or to degradation of mature collagen. We then tested the effect of three distinct PEMF waveforms, two orthogonal coil orientations, and two waveform amplitude levels on the amount of alpha1(I) collagen in the conditioned medium. A sequence of factorial ANOVAs and stepwise regression modeling revealed that the period (duration) of the individual pulses accounted for a significant proportion of the variance associated with the amount of alpha1(I) collagen in the conditioned medium. The total variance accounted for, however, was small (R(2)=0.155, p<0.001 and R(2)=0.172, p<0.001, in the horizontal and vertical orientations, respectively). The positive and negative regression coefficients for the coil orientations revealed that the influence of pulse period was significantly different for the orthogonal coil orientations (p<0.001). The findings imply that the dominant influence of PEMF on the amount of mature, alpha1(I) collagen in the ECM is related to variables other than those expressed in the time-amplitude domain. The results provide objective direction toward identifying waveform characteristics that contribute to the observed between-waveform differences with regard to collagen. Advances in this area may lead toward improving waveforms and waveform delivery protocols.

In vivo longitudinal assessment of bone resorption in a fibular osteotomy model using micro-computed tomography.

Longitudinal in vivo micro-computerized tomography (CT) imaging was used to monitor bone resorption in a rat fibula osteotomy model. Quantitative image post-processing techniques were developed for spatially aligning the longitudinal data sets. Nominal length and bone volume in the proximal and distal segments of the fibula after the osteotomy were measured, and quantitative comparisons of bone loss over a 13-week period post-surgery were made in five individual rats. A significant decrease in nominal length and bone volume of the distal segment was observed 13 weeks post surgery. A significant decrease in bone volume was also observed in the proximal segment. However, no change in nominal length was observed for the proximal segment of the fibula. This study illustrates the power of this non-invasive technology to measure in vivo small changes in bone length and volume using just a small cohort of animals.

Pulsed electromagnetic field treatments enhance the healing of fibular osteotomies.

This study tested the hypothesis that pulsed electromagnetic field (PEMF) treatments augment and accelerate the healing of bone trauma. It utilized micro-computed tomography imaging of live rats that had received bilateral 0.2 mm fibular osteotomies (approximately 0.5% acute bone loss) as a means to assess the in vivo rate dynamics of hard callus formation and overall callus volume. Starting 5 days post-surgery, osteotomized right hind limbs were exposed 3 h daily to Physio-Stim PEMF, 7 days a week for up to 5 weeks of treatment. The contralateral hind limbs served as sham-treated, within-animal internal controls. Although both PEMF- and sham-treatment groups exhibited similar onset of hard callus at approximately 9 days after surgery, a 2-fold faster rate of hard callus formation was observed thereafter in PEMF-treated limbs, yielding a 2-fold increase in callus volume by 13-20 days after surgery. The quantity of the new woven bone tissue within the osteotomy sites was significantly better in PEMF-treated versus sham-treated fibulae as assessed via hard tissue histology. The apparent modulus of each callus was assessed via a cantilever bend test and indicated a 2-fold increase in callus stiffness in the PEMF-treated over sham-treated fibulae. PEMF-treated fibulae exhibited an apparent modulus at the end of 5-weeks that was approximately 80% that of unoperated fibulae. Overall, these data indicate that Physio-Stim PEMF treatment improved osteotomy repair. These beneficial effects on bone healing were not observed when a different PEMF waveform, Osteo-Stim, was used. This latter observation demonstrates the specificity in the relationship between waveform characteristics and biological outcomes.

Bone mass is preserved in a critical-sized osteotomy by low energy pulsed electromagnetic fields as quantitated by in vivo micro-computed tomography.

The effectiveness of non-invasive pulsed electromagnetic fields (PEMF) on stimulating bone formation in vivo to augment fracture healing is still controversial, largely because of technical ambiguities in data interpretation within several previous studies. To address this uncertainty, we implemented a rigorously controlled, blinded protocol using a bilateral, mid-diaphyseal fibular osteotomy model in aged rats that achieved a non-union status within 3-4 weeks post-surgery. Bilateral osteotomies allowed delivery of a PEMF treatment protocol on one hind limb, with the contralateral limb representing a within-animal sham-treatment. Bone volumes in both PEMF-treated and sham-treated fibulae were assessed simultaneously in vivo using highly sensitive, high-resolution micro-computed tomography (microCT) over the course of treatment. We found a significant reduction in the amount of time-dependent bone volume loss in PEMF-treated, distal fibular segments as compared to their contralateral sham-treated bones. Osteotomy gap size was significantly smaller in hind limbs exposed to PEMF over sham-treatment. Therefore, our data demonstrate measurable biological consequences of PEMF exposure on in vivo bone tissue.

K-ras regulates the steady-state expression of matrix metalloproteinase 2 in fibroblasts.

Fibroblasts constitutively express matrix metalloproteinase 2 (MMP-2), which specifically cleaves type IV collagen, a major structural component of basement membranes. The level of MMP-2 expression was not altered by serum withdrawal, suggesting that MMP-2 expression is regulated by a series of steady-state conditions that impinge on the MMP-2 promoter. Expression of a dominant-negative Ras protein significantly inhibited MMP-2 transcription, thereby suggesting a role for steady-state Ras function in the regulation of MMP-2 expression. Kirsten-Ras (K-Ras) knockout fibroblasts express undetectable basal levels of MMP-2, whereas N-Ras knockout fibroblasts expressed constitutive levels of MMP-2 similar to those observed in wild-type control fibroblasts. Using an MMP-2 promoter-luciferase reporter assay, we demonstrated that the transcription of MMP-2 in K-Ras knockout fibroblasts was partially restored by transient expression of c-K(B)-Ras but not c-K(A)-Ras. A phosphoinositide-3 (PI-3) kinase-specific inhibitor (LY294002) decreased the basal level of MMP-2 in wild-type fibroblasts. Blocking PI-3 kinase signaling by overexpression of the regulatory domain of PI-3 kinase (p85) also down-regulated the steady-state MMP-2 levels. Fibroblasts that fail to express AKT1 also expressed decreased amounts of MMP-2 compared with wild-type fibroblasts. These data suggest that steady-state MMP-2 expression is regulated by c-K(B)-Ras through a PI-3 kinase:AKT-dependent signaling pathway. Because the majority of the MMP-2 assays were performed using conditioned media from serum-starved fibroblasts, these data also highlight our previous observations that Ras proteins have functions in the absence of acute mitogenic stimulations. In addition, this is the first demonstration of a specific steady-state function attributable to K(B)-Ras.

Magnetic field visualization in applications to pulsed electromagnetic field stimulation of tissues.

Electromagnetic field visualization is important in multidisciplinary research on the molecular basis of therapeutic effects of pulsed electromagnetic fields (PEMF). We have compared classic PEMF representations by two-dimensional field lines and field magnitude contour plots with a field representation using three-dimensional field isosurfaces. Field simulations were performed for a clinically approved Spinal-Stim Lite system (Orthofix Inc., McKinney, TX). The relatively simple coil system geometry and the predominantly dielectric properties of the surrounding medium (air and human connective tissue) allowed us to develop analytical expressions for the field. The field model was validated by comparison with experimentally measured field values, and with values calculated using a commercial finite-element analysis software package. Two-dimensional field representations by field lines and field contour plots were less intuitive than three-dimensional field isosurface representations to members of the group without an engineering background. Field isosurfaces, represented as three-dimensional solids, allowed for direct visualization of PEMF targeting of individual organs (lumbar spine), the extent of the therapeutic field value, and the directional field characteristics. The dynamic characteristic of the field was well illustrated by a sequence of field isosurfaces corresponding to the evolution with time of the electric current waveform (sawtooth) powering the coils. The isosurface representation of the field can be extended to any three-dimensional coil system geometry using plotting capabilities of current computer algebra software packages.

Cellular N-Ras promotes cell survival by downregulation of Jun N-terminal protein kinase and p38.

Cellular N-Ras provides a steady-state antiapoptotic signal, at least partially through the regulation of phosphorylated Akt and Bad levels. Fibroblasts lacking c-N-Ras expression are highly sensitive to the induction of apoptosis by a variety of agents. Reduction of pBad and pAkt levels using a phosphatidylinositol 3-kinase inhibitor was not sufficient to sensitize the control cell population to the high level of apoptosis observed in the N-Ras knockout cell lines, suggesting that c-N-Ras provides at least one other antiapoptotic signal. Stimulation of the control cells with apoptotic agents results in a transient increase in Jun N-terminal protein kinase (JNK)/p38 activity that decreased to baseline levels during the time course of the experiments. In all cases, however, sustained JNK/p38 activity was observed in cells lacking c-N-Ras expression. This correlated with sustained levels of phosphorylated MKK4 and MKK3/6, upstream activators of JNK and p38, respectively. Mimicking the sustained activation of JNK in the control cells did result in increasing their sensitivity to apoptotic agents, suggesting that prolonged JNK activity is a proapoptotic event. We also examined the potential downstream c-N-Ras targets that might be involved in regulating the duration of the JNK/p38 signal. Only the RalGDS 37G-N-Ras protein protected the N-Ras knockout cells from apoptosis and restored transient rather than sustained JNK activation. These data suggest that cellular N-Ras provides an antiapoptotic signal through at least two distinct mechanisms, one which regulates steady-state pBad and pAkt levels and one which regulates the duration of JNK/p38 activity following an apoptotic challenge.