Synchronous activation of ERK and phosphatidylinositol 3-kinase pathways is required for collagen and extracellular matrix production in keloids

Keloid fibroproliferation appears to be influenced by epithelial-mesenchymal interactions between keloid keratinocytes (KKs) and keloid fibroblasts (KFs). Keloid and normal fibroblasts exhibit accelerated proliferation and collagen I and III production in co-culture with KKs compared with single cell culture or co-culture with normal keratinocytes. ERK and phosphatidylinositol 3-kinase (PI3K) pathway activation has been observed in excessively proliferating KFs in co-culture with KKs. We hypothesized that ERK and PI3K pathways might be involved in collagen and extracellular matrix production in KFs. To test our hypothesis, four samples of KFs were co-cultured in defined serum-free medium with KKs for 2-5 days. KF cell lysate was subjected to Western blot analysis. Compared with KF single cell culture, phospho-ERK1/2 and downstream phospho-Elk-1 showed up-regulation in the co-culture groups, as did phospho-PI3K and phospho-Akt-1, indicating ERK and PI3K pathway activation. Western blotting of the conditioned medium demonstrated increased collagen I-III, laminin beta2, and fibronectin levels. Addition of the MEK1/2-specific inhibitor U0126 or the PI3K-specific inhibitor LY294002 (but not p38 kinase and JNK inhibitors) completely nullified collagen I-III production and significantly decreased laminin beta2 and fibronectin secretion. In the presence of the MEK1/2 or PI3K inhibitor, fibronectin demonstrated changes in molecular mass reflected by faster in-gel migration. These data strongly suggest that synchronous activation of both the ERK and PI3K pathways is essential for collagen I-III and laminin beta2 production. These pathways additionally appear to affect the side chain attachments of fibronectin. Modulation of these pathways may suggest a direction for keloid therapy.     

Kaempferol-induced growth inhibition and apoptosis in A549 lung cancer cells is mediated by activation of MEK-MAPK

A vast variety of naturally occurring substances have been shown to protect against experimental carcinogenesis and an increasing amount of evidence suggests that kaempferol may have cancer chemopreventative properties. However, the precise underlying protective mechanisms are poorly understood. To elucidate these mechanisms, we challenged human lung cancer cell line A549 with kaempferol and investigated its effects upon cellular growth and signal transduction pathways. Treatment of A549 cells with kaempferol resulted in a dose- and time-dependent reduction in cell viability and DNA synthesis with the rate of apoptosis equivalent to 0.9+/-0.5, 5.2+/-1.5, 16.8+/-2.0, 25.4+/-2.6, and 37.8+/-4.5% on treatment with 0, 17.5, 35.0, 52.5, and 70.0 microM kaempferol, respectively. Concomitantly, kaempferol treatments led to a 1.2-, 2.7-, 3.3-, and 3.4-fold increase in Bax. Similar elevations were also observed in Bad which increased 1.2-, 3.3-, 3.7-, and 4.7-fold, respectively, as compared to control. Bcl-2 and Bcl-xL expression were inhibited in a dose-dependent fashion. While the Akt-1 and phosphorylated Akt-1 were inhibited, the mitogen-activated protein kinase (MAPK) was activated upon kaempferol treatment. Kaempferol induced apoptosis was associated with the cleavage of caspase-7 and poly ADP-ribose polymerase (PARP). Inhibition of MEK1/2 but not PI-3 kinase blocked kaempferol-induced cleavage of caspase-7, PARP cleavage, and apoptosis. The results suggest that inactivation of Akt-1 and alteration of Bcl-2 family of proteins are not sufficient for kaempferol to induce apoptosis and activation of MEK-MAPK is a requirement for kaempferol-induced cell death machinery in A549 cells.     

Facile electron transfer during formation of cluster X and kinetic competence of X for tyrosyl radical production in protein R2 of ribonucleotide reductase from mouse.

The kinetics and mechanism of formation of the tyrosyl radical and mu-(oxo)diiron(III) cluster in the R2 subunit of ribonucleotide reductase from mouse have been examined by stopped-flow absorption and freeze-quench electron paramagnetic resonance and M?ssbauer spectroscopies. The reaction comprises (1) acquisition of Fe(II) ions by the R2 apo protein, (2) activation of dioxygen at the resulting carboxylate-bridged diiron(II) cluster to form oxidized intermediate diiron species, and (3) univalent oxidation of Y177 by one of these intermediates to form the stable radical, with concomitant or subsequent formation of the adjacent mu-(oxo)diiron(III) cluster. The data establish that an oxidized diiron intermediate spectroscopically similar to the well-characterized, formally Fe(III)Fe(IV) cluster X from the reaction of the Escherichia coli R2 protein precedes the Y177 radical in the reaction sequence and is probably the Y177 oxidant. As formation of the X intermediate (1) requires transfer of an "extra" reducing equivalent to the buried diiron cluster following the addition of dioxygen and (2) is observed to be rapid relative to other steps in the reaction, the present data indicate that the transfer of this reducing equivalent is not rate-limiting for Y177 radical formation, in contrast to what was previously proposed (Schmidt, P. P., Rova, U., Katterle, B., Thelander, L., and Gr?slund, A. (1998) J. Biol. Chem. 273, 21463-21472). Indeed, the formation of X (k(obs) = 13 +/- 3 s(-1) at 5 degrees C and 0.95 mM O(2)) and the decay of the intermediate to give the Y177 radical (k(obs) = 5 +/- 2 s(-1)) are both considerably faster than the formation of the reactive Fe(II)-R2 complex from the apo protein and Fe(II)(aq) (k(obs) = 0.29 +/- 0.03 s(-1)), which is the slowest step overall. The conclusions that cluster X is an intermediate in Y177 radical formation and that transfer of the reducing equivalent is relatively facile imply that the mouse R2 and E. coli R2 reactions are mechanistically similar.     

M?ssbauer study of cytochrome c2 from Rhodospirillum rubrum. Sign of the product gxgygz of some low spin ferric heme proteins.

We have studied cytochrome c2 from Rhodospirllum rubrum with M?ssbauer spectroscopy and electron paramagnetic resonance. The M?ssbauer data on the ferric protein, taken in external magnetic fields up to 50 kG, were analyzed within the framework of the ligand field model commonly used to evaluate low-spin ferric heme compounds. The data analysis shows that the determinant of the electronic g-tensor, i.e. the product gxgygz, is positive for cytochrome c2. We have reanalyzed published M?ssbauer data of some low-spin ferric heme proteins with respect to the sign of the g-tensor determinant. We find that gxgygz is also positive for the cytochromes c, bs, and P-450, and for chloroperoxidase.     

The biosynthesis of polyunsaturated fatty acids by rat sertoli cells.

1. The biosynthesis of polyunsaturated fatty acids (PUFA) of the n-6 and n-3 series was investigated in cultured Sertoli cells. 18:2n-6, 18:3n-6, 20:2n-6, 18:3n-3 and 20:3n-3 were added individually at a concentration of 20 mumol to culture media. 2. Maximum incorporation of 20- and 22-carbon PUFA into membrane lipids was observed after 72 hr of incubation with all the exogenous substrates used. 3. As reported in other cell systems, the delta 6 desaturation was the first rate-limiting step; the major factor regulating this activity was the concentration of linoleic acid or alpha-linolenic acid in the medium. 4. Our data show that the delta 5-desaturation represents a second regulatory step in PUFA biosynthesis. 5. The sum of n-6 and n-3 PUFA of the 22 carbon chain length constantly represented between 11 and 12% of total fatty acids, regardless of the exogenous substrate used. 6. Our kinetic studies of the incorporation of PUFA of the n-6 and n-3 series did not permit detection of a delta 8 desaturase activity.     

High-resolution visualization of oxygen distribution in the liver in vivo

Microcirculatory failure after stress events results in mismatch in oxygen supply and demand. Determination of tissue oxygen distribution in vivo may help elucidate mechanisms of injury, but present methods have limited resolution. Male Sprague-Dawley rats were anesthetized, prepared for intravital microscopy, and received intravenously the oxygen-sensitive fluorescent dye Tris(1,10-phenanthroline)ruthenium(II) chloride hydrate [Ru(phen)3(2+)]. An impaired hepatic oxygen distribution was induced by either phenylephrine or hemorrhage. Intensity of Ru(phen)3(2+) fluorescence was compared with NADH autofluorescence indicating changes in the mitochondrial redox potential. Ethanol was injected to affect the NADH-to-NAD+ ratio without altering the P(O2). Infusion of Ru(phen)3(2+) resulted in a heterogeneous fluorescence under baseline conditions reflecting the physiological acinar P(O2) distribution. A decrease in oxygen supply due to phenylephrine or hemorrhage was paralleled by an increase in Ru(phen)3(2+) and NADH fluorescence reflecting an impaired mitochondrial redox state. Ethanol did not alter Ru(phen)3(2+) fluorescence but increased NADH fluorescence indicating independence of P(O2) and redox state imaging. Intravenous administration of Ru(phen)3(2+) for intravital videomicroscopy represents a new method to visualize the hepatic tissue P(O2). Combined with NADH autofluorescence, it provides additional information regarding the tissue redox state.