Rational Engineering of 3α-Hydroxysteroid Dehydrogenase/Carbonyl Reductase for a Biomimetic Nicotinamide Mononucleotide Cofactor

Enzymes are powerful biological catalysts for natural substrates but they have low catalytic efficiency for non-natural substrates. Protein engineering can be used to optimize enzymes for catalysis and stability. 3α-Hydroxysteroid dehydrogenase/carbonyl reductase (3α-HSD/CR) catalyzes the oxidoreduction reaction of NAD+ with androsterone. Based on the structure and catalytic mechanism, we mutated the residues of T11, I13, D41, A70, and I112 and they interacted with different portions of NAD+ to switch cofactor specificity to biomimetic cofactor nicotinamide mononucleotide (NMN+). Compared to wild-type 3α-HSD/CR, the catalytic efficiency of these mutants for NAD+ decreased significantly except for the T11 mutants but changed slightly for NMN+ except for the A70K mutant. The A70K mutant increased the catalytic efficiency for NMN+ by 8.7-fold, concomitant with a significant decrease in NAD+ by 1.4 × 104-fold, resulting in 9.6 × 104-fold cofactor specificity switch toward NMN+ over NAD+. Meanwhile, the I112K variant increased the thermal stability and changed to a three-state transition from a two-state transition of thermal unfolding of wild-type 3α-HSD/CR by differential scanning fluorimetry. Molecular docking analysis indicated that mutations on these residues affect the position and conformation of the docked NAD+ and NMN+, thereby affecting their activity. A70K variant sterically blocks the binding with NAD+, restores the H-bonding interactions of catalytic residues of Y155 and K159 with NMN+, and enhances the catalytic efficiency for NMN+

Ethanol May Suppress Wnt/β-catenin Signaling on Human Bone Marrow Stroma Cells: A Preliminary Study

Ethanol and glucocorticoids are risk factors associated with osteonecrosis. Previous reports suggest ethanol and glucocorticoids induce adipogenesis, decrease osteogenesis in bone marrow stroma cells, and produce intracellular lipid deposits resulting in death of osteocytes. The Wnt/β-catenin signal pathway is involved in the regulation of homeostasis of bone and we presume glucocorticoids and ethanol may induce osteonecrosis in humans through a similar mechanism as in rodents. We hypothesized (1) ethanol, like glucocorticoids, decreases osteogenesis and increases adipogenesis through the Wnt/β-catenin signaling pathway in human bone marrow stromal cells; and (2) ethanol decreases intranuclear translocation of β-catenin. We found both dexamethasone and ethanol decrease the gene and protein expression of osteogenesis and increase that of adipogenesis through Wnt signaling-related genes by semiquantitative and quantitative polymerase chain reaction and Western blot. Ethanol hampered intranuclear translocation of β-catenin by immunofluorescence analysis. The data suggest the Wnt/β-catenin signaling pathway may be associated with ethanol-induced osteonecrosis

Low-Power GaAlAs Laser Irradiation Promotes the Proliferation and Osteogenic Differentiation of Stem Cells via IGF1 and BMP2

<div><p>Low-power laser irradiation (LPLI) has been found to induce various biological effects and cellular processes. Also, LPLI has been shown to promote fracture repair. Until now, it has been unclear how LPLI promotes bone formation and fracture healing. The aim of this study was to investigate the potential mechanism of LPLI-mediated enhancement of bone formation using mouse bone marrow mesenchymal stem cells (D1 cells). D1 cells were irradiated daily with a gallium-aluminum-arsenide (GaAlAs) laser at dose of 0, 1, 2, or 4 J/cm². The lactate dehydrogenase (LDH) assay showed no cytotoxic effects of LPLI on D1 cells, and instead, LPLI at 4 J/cm² significantly promoted D1 cell proliferation. LPLI also enhanced osteogenic differentiation in a dose-dependent manner and moderately increased expression of osteogenic markers. The neutralization experiments indicated that LPLI regulated insulin-like growth factor 1 (IGF1) and bone morphogenetic protein 2 (BMP2) signaling to promote cell proliferation and/or osteogenic differentiation. In conclusion, our study suggests that LPLI may induce IGF1 expression to promote both the proliferation and osteogenic differentiation of D1 cells, whereas it may induce BMP2 expression primarily to enhance osteogenic differentiation.</p> </div

LPLI enhances the proliferation of D1 cells.

<p>Cells were treated with LPLI at doses of 0 (control), 1, 2, or 4 J/cm² and assayed at days 1, 3 and 5. (<b>A</b>) LDH leakage was analyzed to evaluate cell cytotoxicity. There were no significant differences between the groups (n = 12). (<b>B</b>) Cell numbers were counted at indicated times. There were no differences between the cell numbers of each group at day 1 and day 3. At day 5, the cell numbers of the LPLI groups treated with 2 and 4 J/cm² were significantly higher than that of control group (n = 6). (<b>C</b>) An MTT assay was performed, and the optical densities were measured. Similar results with the cell counting analysis was found (n = 18). The statistical significance levels were as follows: * p<0.05, ** p<0.01, and *** p<0.001 compared with the control group.</p

BMP2 regulates LPLI-mediated osteogenic differentiation, but not cellular proliferation.

<p>(<b>A</b>) D1 cells were treated with LPLI at doses of 0 (control) or 4 J/cm². BMP2 protein levels in conditioned culture medium on day 5 were determined by ELISA. LPLI significantly increased the BMP2 protein levels in culture medium (n = 8). (<b>B</b>) D1 cells were irradiated with or without LPLI (4 J/cm²) and cultured with a BMP2 neutralizing antibody (anti-BMP2, 10 µg/ml) or the same amount of a control antibody (anti-IgG2a, 10 µg/ml). After culture for 5 days, cell viability was determined by an MTT assay. The proliferative activity was significantly decreased in the control cells, but not in the LPLI-stimulated cells (n = 18). (<b>C</b>) Cells underwent a similar treatment as described in B, except that D1 cells were cultured in OIM for 10 days. The relative osteogenic activity was quantified using Alizarin Red S staining. Anti-BMP2 neutralizing antibodies significantly decreased mineral deposition in the control and the LPLI-stimulated cells (n = 6). The following statistical levels were applied: * p<0.05, ** p<0.01 and *** p<0.001.</p

IGF1 regulated LPLI-mediated proliferation and osteogenic differentiation of D1 cells.

<p>(<b>A</b>) D1 cells were treated with LPLI at doses of 0 (control) or 4 J/cm². IGF1 protein levels in conditioned culture medium on day 5 were determined by ELISA. LPLI significantly increased the IGF1 protein levels in culture medium (n = 8). (<b>B</b>) D1 cells were irradiated with or without LPLI (4 J/cm²) and cultured with an IGF1 neutralizing antibody (anti-IGF1, 10 µg/ml) or the same amount of a control antibody (anti-IgG2a, 10 µg/ml). After culture for 5 days, cell viability was determined by an MTT assay. The proliferation of D1 cells was significantly decreased in the control and the LPLI-stimulated cells by blocking with IGF1 antibody (n = 18). (<b>C</b>) Cells underwent a similar treatment as described in B, except that D1 cells were cultured in OIM for 10 days. The relative osteogenic activity was quantified using Alizarin Red S staining. The mineralization was significantly inhibited by treatment with anti-IGF1 antibodies in the control and the LPLI-stimulated cells (n = 6). The following statistical levels were applied: * p<0.05 and ** p<0.01.</p