Chi-Ching Hwang

Advanced glycation end‐products activate extracellular signal‐regulated kinase via the oxidative stress‐EGF receptor pathway in renal fibroblasts

Advanced glycation end‐products (AGEs), epidermal growth factor receptor (EGFR), reactive oxygen species (ROS), and extracellular signal‐regulated kinases (ERK) are implicated in diabetic nephropathy (DN). Therefore, we asked if AGEs‐induced ERK protein phosphorylation and mitogenesis are dependent on the receptor for AGEs (RAGE)–ROS–EGFR pathway in normal rat kidney interstitial fibroblast (NRK‐49F) cells. We found that AGEs (100 µg/ml) activated EGFR and ERK1/2, which was attenuated by RAGE short‐hairpin RNA (shRNA). AGEs also increased RAGE protein and intracellular ROS levels while RAGE shRNA and N‐acetylcysteine (NAC) attenuated AGEs‐induced intracellular ROS. Hydrogen peroxide (5–25 µM) increased RAGE protein level while activating both EGFR and ERK1/2. Low‐dose hydrogen peroxide (5 µM) increased whereas high‐dose hydrogen peroxide (100 µM) decreased mitogenesis at 3 days. AGEs‐activated EGFR and ERK1/2 were attenuated by an anti‐oxidant (NAC) and an EGFR inhibitor (Iressa). Moreover, AGEs‐induced mitogenesis was attenuated by RAGE shRNA, NAC, Iressa, and an ERK1/2 inhibitor (PD98059). In conclusion, it was found that AGEs‐induced mitogenesis is dependent on the RAGE–ROS–EGFR–ERK1/2 pathway whereas AGEs‐activated ERK1/2 is dependent on the RAGE–ROS–EGFR pathway in NRK‐49F cells. J. Cell. Biochem. 109: 38–48, 2010.

Mechanism of Proton Transfer in the 3α-Hydroxysteroid Dehydrogenase/Carbonyl Reductase from Comamonas testosteroni

3α-Hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni catalyzes the oxidation of androsterone with NAD+ to form androstanedione and NADH with a concomitant releasing of protons to bulk solvent. To probe the proton transfer during the enzyme reaction, we used mutagenesis, chemical rescue, and kinetic isotope effects to investigate the release of protons. The kinetic isotope effects of DV and D2OV for wild-type enzyme are 1 and 2.1 at pL 10.4 (where L represents H, 2H), respectively, and suggest a rate-limiting step in the intramolecular proton transfer. Substitution of alanine for Lys159 changes the rate-limiting step to the hydride transfer, evidenced by an equal deuterium isotope effect of 1.8 on Vmax and V/Kandrosterone and no solvent kinetic isotope effect at saturating 3-(cyclohexylamino)propanesulfonic acid (CAPS). However, a value of 4.4 on Vmax is observed at 10 mm CAPS at pL 10.4, indicating a rate-limiting proton transfer. The rate of the proton transfer is blocked in the K159A and K159M mutants but can be rescued using exogenous proton acceptors, such as buffers, small primary amines, and azide. The Brønsted relationship between the log(V/Kd-baseEt) of the external amine (corrected for molecular size effects) and pKa is linear for the K159A mutant-catalyzed reaction at pH 10.4 (β = 0.85 ± 0.09) at 5 mm CAPS. These results show that proton transfer to the external base with a late transition state occurred in a rate-limiting step. Furthermore, a proton inventory on V/Et is bowl-shaped for both the wild-type and K159A mutant enzymes and indicates a two-proton transfer in the transition state from Tyr155 to Lys159 via 2′-OH of ribose.

GSKIP, an inhibitor of GSK3β, mediates the N-cadherin/β-catenin pool in the differentiation of SH-SY5Y cells†

Emerging evidence has shown that GSK3β plays a pivotal role in regulating the specification of axons and dendrites. Our previous study has shown a novel GSK3β interaction protein (GSKIP) able to negatively regulate GSK3β in Wnt signaling pathway. To further characterize how GSKIP functions in neurons, human neuroblastoma SH‐SY5Y cells treated with retinoic acid (RA) to differentiate to neuron‐like cells was used as a model. Overexpression of GSKIP prevents neurite outgrowth in SH‐SY5Y cells. GSKIP may affect GSK3β activity on neurite outgrowth by inhibiting the specific phosphorylation of tau (ser396). GSKIP also increases β‐catenin in the nucleus and raises the level of cyclin D1 to promote cell‐cycle progression in SH‐SY5Y cells. Additionally, overexpression of GSKIP downregulates N‐cadherin expression, resulting in decreased recruitment of β‐catenin. Moreover, depletion of β‐catenin by small interfering RNA, neurite outgrowth is blocked in SH‐SY5Y cells. Altogether, we propose a model to show that GSKIP regulates the functional interplay of the GSK3β/β‐catenin, β‐catenin/cyclin D1, and β‐catenin/N‐cadherin pool during RA signaling in SH‐SY5Y cells. J. Cell. Biochem. 108: 1325–1336, 2009.

Arecoline-induced phosphorylated p53 and p21WAF1 protein expression is dependent on ATM/ATR and phosphatidylinositol-3-kinase in clone-9 cells

Betel‐quid use is associated with liver cancer whereas its constituent arecoline is cytotoxic, genotoxic, and induces p53‐dependent p21WAF1 protein expression in Clone‐9 cells (rat hepatocytes). The ataxia telangiectasia mutated (ATM)/rad3‐related (ATR)‐p53‐p21WAF1 and the phosphatidylinositol‐3‐kinase (PI3K)‐mammalian target of rapamycin (mTOR) pathways are involved in the DNA damage response and the pathogenesis of cancers. Thus, we studied the role of ATM/ATR and PI3K in arecoline‐induced p53 and p21WAF1 protein expression in Clone‐9 cells. We found that arecoline (0.5 mM) activated the ATM/ATR kinase at 30 min. The arecoline‐activated ATM/ATR substrate contained p‐p53Ser15. Moreover, arecoline only increased the levels of the p‐p53Ser6, p‐p53Ser15, and p‐p53Ser392 phosphorylated p53 isoforms among the known isoforms. ATM shRNA attenuated arecoline‐induced p‐p53Ser15 and p21WAF1 at 24 h. Arecoline (0.5 mM) increased phosphorylation levels of p‐AktSer473 and p‐mTORSer2448 at 30–60 min. Dominant‐negative PI3K plasmids attenuated arecoline‐induced p21WAF1, but not p‐p53Ser15, at 24 h. Rapamycin attenuated arecoline‐induced phosphrylated p‐p53Ser15, but not p21WAF1, at 24 h. ATM shRNA, but not dominant‐negative PI3K plasmids, attenuated arecoline‐induced p21WAF1 gene transcription. We conclude that arecoline activates the ATM/ATR‐p53‐p21WAF1 and the PI3K/Akt‐mTOR‐p53 pathways in Clone‐9 cells. Arecoline‐induced phosphorylated p‐p53Ser15 expression is dependent on ATM whereas arecoline‐induced p21WAF1 protein expression is dependent on ATM and PI3K. Moreover, p21WAF1 gene is transcriptionally induced by arecoline‐activated ATM. J. Cell. Biochem. 107: 408–417, 2009.

Mechanism of proton transfer in the 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni.

3α-Hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni catalyzes the oxidation of androsterone with NAD+ to form androstanedione and NADH with a concomitant releasing of protons to bulk solvent. To probe the proton transfer during the enzyme reaction, we used mutagenesis, chemical rescue, and kinetic isotope effects to investigate the release of protons. The kinetic isotope effects of DV and D2OV for wild-type enzyme are 1 and 2.1 at pL 10.4 (where L represents H, 2H), respectively, and suggest a rate-limiting step in the intramolecular proton transfer. Substitution of alanine for Lys159 changes the rate-limiting step to the hydride transfer, evidenced by an equal deuterium isotope effect of 1.8 on Vmax and V/Kandrosterone and no solvent kinetic isotope effect at saturating 3-(cyclohexylamino)propanesulfonic acid (CAPS). However, a value of 4.4 on Vmax is observed at 10 mm CAPS at pL 10.4, indicating a rate-limiting proton transfer. The rate of the proton transfer is blocked in the K159A and K159M mutants but can be rescued using exogenous proton acceptors, such as buffers, small primary amines, and azide. The Brønsted relationship between the log(V/Kd-baseEt) of the external amine (corrected for molecular size effects) and pKa is linear for the K159A mutant-catalyzed reaction at pH 10.4 (β = 0.85 ± 0.09) at 5 mm CAPS. These results show that proton transfer to the external base with a late transition state occurred in a rate-limiting step. Furthermore, a proton inventory on V/Et is bowl-shaped for both the wild-type and K159A mutant enzymes and indicates a two-proton transfer in the transition state from Tyr155 to Lys159 via 2′-OH of ribose.

Advanced glycation end-product-inhibited cell proliferation and protein expression of β-catenin and cyclin D1 are dependent on glycogen synthase kinase 3β in LLC-PK1 cells

Glycogen synthase kinase 3beta (GSK3beta) is increased by high glucose in mesangial cells. Thus, we studied the role of GSK3beta in advanced glycation end-product (AGE)-induced effects in the proximal tubule-like LLC-PK1 cells. We found that AGE (100 microg/ml) time-dependently (8-48 h) increased phospho-GSK3beta-Tyr216 (active GSK3beta) and time-dependently (4-24 h) decreased phospho-GSK3beta-Ser21/9 (inactive GSK3beta) protein expression. Meanwhile, AGE (100 microg/ml) activated GSK3beta kinase at 8-48 h. AGE (100 microg/ml) dose-dependently (75-100 microg/ml) decreased beta-catenin protein expression but AGE did not decrease beta-catenin protein expression until 48 h. SB216763 (a GSK3beta inhibitor) and GSK3beta shRNA attenuated AGE (100 microg/ml)-inhibited cell proliferation and protein expression of beta-catenin and cyclin D1 at 48 h. SB216763 also attenuated AGE-induced type IV collagen. We conclude that AGE activates GSK3beta in LLC-PK1 cells. AGE-inhibited beta-catenin and cyclin D1 protein expression are dependent on GSK3beta. Moreover, AGE-inhibited cell proliferation and AGE-induced type IV collagen protein expression are dependent on GSK3beta.

Role of S114 in the NADH-induced conformational change and catalysis of 3α-hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni

3alpha-Hydroxysteroid dehydrogenase/carbonyl reductase reversibly catalyzes the oxidation of androsterone with NAD(+) to form androstanedione and NADH. In this study, we characterize the role of the conserved residue S114 in cofactor binding and catalysis, using site-directed mutagenesis, steady-state kinetics, fluorescence quenching and anisotropy measurements. The catalytic efficiency of V/K(NADH)Et for wild-type and S114A is 1.5 x10(7) and 3.8 x 10(3) M(-1) s(-1), respectively, suggesting that NADH association to wild-type and S114A mutant enzymes involves two steps, a bimolecular binding step and isomerization. The binding of NADH into a hydrophobic pocket in the active site of wild-type and S114A mutant enzymes restricts its motion and shields the fluorescence quenching from solvent, with an increase in the fluorescence intensity and a blue shift at the maximum wavelength. Furthermore, the binding of NADH leads to the protein fluorescence quenching, mainly due to fluorescence resonance energy transfer to NADH. S114A mutant enzyme decreases 3100-fold in V/Et with no apparent change in K(m) for substrates. Addition of NADH to S114A mutant enzyme induces a secondary structural change. These results suggest that S114 is important to maintain the correct conformation for the nucleotide binding and facilitate the reaction. Substitution of alanine for S114 eliminates the hydrogen bonding interaction with P185, causing a conformational change in a nonproductive binding of NADH and a significant loss of activity.

Mechanism of Proton Transfer in the 3 -Hydroxysteroid Dehydrogenase/Carbonyl Reductase from Comamonas testosteroni

3α-Hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni catalyzes the oxidation of androsterone with NAD+ to form androstanedione and NADH with a concomitant releasing of protons to bulk solvent. To probe the proton transfer during the enzyme reaction, we used mutagenesis, chemical rescue, and kinetic isotope effects to investigate the release of protons. The kinetic isotope effects of DV and D2OV for wild-type enzyme are 1 and 2.1 at pL 10.4 (where L represents H, 2H), respectively, and suggest a rate-limiting step in the intramolecular proton transfer. Substitution of alanine for Lys159 changes the rate-limiting step to the hydride transfer, evidenced by an equal deuterium isotope effect of 1.8 on Vmax and V/Kandrosterone and no solvent kinetic isotope effect at saturating 3-(cyclohexylamino)propanesulfonic acid (CAPS). However, a value of 4.4 on Vmax is observed at 10 mm CAPS at pL 10.4, indicating a rate-limiting proton transfer. The rate of the proton transfer is blocked in the K159A and K159M mutants but can be rescued using exogenous proton acceptors, such as buffers, small primary amines, and azide. The Brønsted relationship between the log(V/Kd-baseEt) of the external amine (corrected for molecular size effects) and pKa is linear for the K159A mutant-catalyzed reaction at pH 10.4 (β = 0.85 ± 0.09) at 5 mm CAPS. These results show that proton transfer to the external base with a late transition state occurred in a rate-limiting step. Furthermore, a proton inventory on V/Et is bowl-shaped for both the wild-type and K159A mutant enzymes and indicates a two-proton transfer in the transition state from Tyr155 to Lys159 via 2′-OH of ribose.

Contributions of active site residues to cofactor binding and catalysis of 3α-hydroxysteroid dehydrogenase/carbonyl reductase

3alpha-Hydroxysteroid dehydrogenase/carbonyl reductase reversely catalyzes the oxidation of androsterone with NAD(+) to form androstanedione and NADH. In this study, we investigated the function of active site residues N86, Y155, and K159 in NADH binding and catalysis in the reduction of androstanedione, using site-directed mutagenesis, steady-state kinetics, fluorescence quenching, and anisotropy measurements. The N86A, Y155F, and K159A mutant enzymes decreased the catalytic constant by 37- to 220-fold and increased the dissociation constant by 3- to 75-fold, respectively. Binding of NADH with wild-type and mutant enzymes caused different levels of fluorescence resonance energy transfer, implying a different orientation of nicotinamide ring versus W173. In addition, the enzyme-bound NADH decreased the fluorescence anisotropy value in the order WT>N86A>Y155F>K159A, indicating an increase in the mobility of the bound NADH for the mutants. Data suggest that hydrogen bonding with the hydroxyl group of nicotinamide ribose by K159 and Y155 is important to maintain the orientation of NADH and contributes greatly to the transition-state binding energy to facilitate the catalysis. N86 is important for stabilizing the position of K159. Substitution of alanine for N86 has a minor effect on NADH binding through K159, resulting in a slight increase in the mobility of the bound NADH and decreases in affinity and catalytic constant.

Interactions across the interface contribute the stability of homodimeric 3α-hydroxysteroid dehydrogenase/carbonyl reductase

The dimerization of 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase was studied by interrupting the salt bridge interactions between D249 and R167 in the dimeric interface. Substitution of alanine, lysine and serine for D249 decreased catalytic efficiency 30, 1400 and 1.4-fold, and lowered the melting temperature 6.9, 5.4 and 7.6 degrees C, respectively. The mutated enzymes have the dimeric species but the equilibrium between monomer and dimer for these mutants varies from each other, implying that these residues might contribute differently to the dimer stability. Thermal and urea-induced unfolding profiles for wild-type and mutant enzymes appeared as a two-state transition and three-state transition, respectively. In addition, mutation on D249 breaks the salt bridges and causes different effects on the loss of enzymatic activity for D249A, D249K and D249S mutants in the urea-induced unfolding profiles. Hence, D249 at the dimeric interface in 3alpha-HSD/CR is essential for conformational stability, oligomeric integrity and enzymatic activity.