ABSTRACT
Seed dormancy and germination determine the beginning of the life cycle of plants, and the phytohormone ABA plays a crucial role in regulation of seed dormancy and germination. However, the upstream regulatory mechanism of ABA metabolism during dormancy releasing is still remain elusive. In this paper, we present a novel mechanism of OsNAC2 in controlling ABA metabolism and regulation of seed dormancy. OsNAC2 highly expressed during seed development and germination, and overexpression of OsNAC2 strengthened seed dormancy and suppressed germination. Moreover, exogenous phytohormone treatment showed that OsNAC2 acted upstream of GA signaling and downstream of ABA signaling. Additionally, overexpression of OsNAC2 inhibited ABA degradation and increased ABA content during early germination. Further molecular analysis revealed that OsNAC2 directly bound to the ABA metabolism genes promoter and inhibits their transcription in rice protoplasts. These finding could help us explain the genetic regulation mechanism of ABA metabolism during dormancy release and germination in rice.
Subject(s)
Oryza , Plant Dormancy , Plant Dormancy/genetics , Plant Growth Regulators/pharmacology , Plant Growth Regulators/metabolism , Oryza/genetics , Oryza/metabolism , Abscisic Acid/pharmacology , Abscisic Acid/metabolism , Germination/genetics , Seeds/metabolism , Gene Expression Regulation, PlantABSTRACT
Parathyroid hormone (PTH) and its related peptide (PTH-related peptide 1-34) are two of the Food and Drug Administration-approved bone-promoting drugs for age-related osteoporosis. Treatment with PTH stimulates bone formation. However, the molecular mechanisms of PTH-mediated osteoblast differentiation and cell proliferation are still not completely understood. In this study, we showed that PTH induced endoplasmic reticulum (ER) stress in osteoblasts through the PKR-like endoplasmic reticulum kinase (PERK)-eukaryotic initiation factor 2α (EIF2α)-activating transcription factor 4 (ATF4)-signaling pathway. After separately blocking PERK-EIF2α-ATF4 signaling with two different inhibitors [AMG'44 and integrated stress response inhibitor (ISRIB)] or specific small interfering RNA for PERK and ATF4, the following targets were all downregulated: expression of osteoblast differentiation markers [runt-related transcription factor 2 (Runx2), alkaline phosphatase (Alp), type I collagen (Col1a1), and osteocalcin (Ocn)], cell proliferation markers (CyclinE, CyclinD, and CDC2), amino acid import (Glyt1), and metabolism-related genes (Asns). Additionally, Alp-positive staining cells, Alp activity, matrix mineralization, Ocn secretion, and cell proliferation indexes were inhibited. Interestingly, we found that salubrinal enhanced PTH-induced osteoblast differentiation and proliferation by maintenance of phosphorylation of EIF2α. Furthermore, we observed that PTH increased the association between heat shock protein 90 (HSP90) and PERK and maintained PERK protein stabilization in the early stages of PTH-induced ER stress. Treatment of MC3T3-E1 cells with geldanamycin, an HSP90 inhibitor, decreased PERK protein expression and inhibited osteoblast differentiation and cell proliferation upon PTH treatment. Taken together, our data demonstrate that PTH regulates osteoblast differentiation and cell proliferation, partly by activating the HSP90-dependent PERK-EIF2α-ATF4 signaling pathway.
Subject(s)
Cell Differentiation/drug effects , Cell Proliferation/drug effects , Endoplasmic Reticulum Stress/drug effects , Osteoblasts/drug effects , Parathyroid Hormone/pharmacology , Activating Transcription Factor 4/metabolism , Alkaline Phosphatase/drug effects , Alkaline Phosphatase/metabolism , Animals , Benzoquinones/pharmacology , CDC2 Protein Kinase/drug effects , CDC2 Protein Kinase/metabolism , Cell Line , Collagen Type I/drug effects , Collagen Type I/metabolism , Collagen Type I, alpha 1 Chain , Core Binding Factor Alpha 1 Subunit/drug effects , Core Binding Factor Alpha 1 Subunit/metabolism , Cyclin D/drug effects , Cyclin D/metabolism , Cyclin E/drug effects , Cyclin E/metabolism , Enzyme Inhibitors/pharmacology , Eukaryotic Initiation Factor-2/metabolism , Glycine Plasma Membrane Transport Proteins/drug effects , Glycine Plasma Membrane Transport Proteins/metabolism , HSP90 Heat-Shock Proteins/antagonists & inhibitors , HSP90 Heat-Shock Proteins/drug effects , HSP90 Heat-Shock Proteins/metabolism , Lactams, Macrocyclic/pharmacology , Mice , Osteoblasts/metabolism , Osteocalcin/drug effects , Osteocalcin/metabolism , Signal Transduction , eIF-2 Kinase/metabolismABSTRACT
Osteoblast uses aerobic glycolysis to meet the metabolic needs in differentiation process. Lactate, the end product of glycolysis, presents in the environment with elevated PTH and osteoblast differentiation. Although previous findings showed that lactate promoted osteoblast differentiation, whether lactate affects PTH-mediated osteoblast differentiation is unclear. To investigate this, pre-osteoblast cell line MC3T3-E1 was treated PTH with or without physiological dose of lactate. Lactate increases ALP positive cell formation, increases ALP activity and expression of differentiation related markers, enriches the CREB transcriptional factor target genes in PTH treated cells. Using inhibitors for MCT-1 reveales that lactate effects are MCT-1 independent. Lactate selectively increases Akt and p38 activation but not Erk1/2 and ß-Catenin activation. The inhibitors for Akt and p38 inhibit lactate effects on PTH mediated osteoblast differentiation. Using inhibitors for Gαi signaling of GPR81 further increases Alp mRNA levels in lactate and PTH co-treatment cells. However, with the inhibitors for Gßγ-PLC-PKC signaling, the effect of lactate on PTH mediated osteoblast differentiation is inhibited. Our data demonstrate that lactate activates GPR81-Gßγ-PLC-PKC-Akt signaling to regulate osteoblast differentiation that mediated by PTH treatment.
