ABSTRACT
The transcriptional cofactor CITED1 inhibits osteoblastic differentiation and blunts the stimulation of osteoblastic differentiation by parathyroid hormone (PTH). In the MC3T3-E1 osteoblastic cell line, we found that CITED1 was located predominantly in the cytoplasm and that hPTH(1-34) increased translocation of CITED1 from the cytoplasm to the nucleus. This response to hPTH(1-34) was not observed when all 9 serine residues within the 63-84 domain of CITED1 were mutated to alanines (CITED1 9S>A) or when a single serine to alanine mutation was made at position 79 (CITED1 S(79)>A). CITED1 containing mutations of these 9 serines to glutamic acid (9S>E) retained the same nuclear translocation response to hPTH(1-34) as the wild type CITED1. ALP activity and formation of mineralized nodules were inhibited in cells transfected with pcDNA3-CFP-CITED1 or with pcDNA3-CFP-CITED1 9S>E with or without hPTH(1-34) treatment (all P<0.05); these changes were not observed using CITED1 9S>A. Cells exposed to intermittent treatment with hPTH(1-34) expressed more ALP2, Runx2 and osteocalcin than vehicle-treated cells. These effects of hPTH(1-34) were inhibited in cells transfected with pcDNA3-CFP-CITED1 or pcDNA3-CFP-CITED1 9S>E, but were slightly enhanced by the alanine mutants. PKC activator (TPA) increased nuclear translocation of CITED1, whereas a PKC inhibitor (Go6983) blunted the effect of hPTH(1-34) on the nuclear translocation of wildtype CITED1 but not of CITED1 S(79)>E. The data indicated that serine phosphorylation at position 79 in the 63-84 domain is associated with PKC activation, and is required for both CITED1 nuclear translocation induced by PTH and the negative effects of CITED1 on osteoblastic differentiation and mineralization.
Subject(s)
Calcification, Physiologic/physiology , Cell Differentiation/physiology , Cell Nucleus/metabolism , Nuclear Proteins/metabolism , Osteoblasts/metabolism , Parathyroid Hormone/pharmacology , Trans-Activators/metabolism , Active Transport, Cell Nucleus/drug effects , Active Transport, Cell Nucleus/physiology , Amino Acid Substitution , Animals , Apoptosis Regulatory Proteins , Calcification, Physiologic/drug effects , Cell Differentiation/drug effects , Cell Line , Cell Nucleus/genetics , Core Binding Factor Alpha 1 Subunit/genetics , Core Binding Factor Alpha 1 Subunit/metabolism , Humans , Indoles/pharmacology , Maleimides/pharmacology , Mice , Mutation, Missense , Nuclear Proteins/genetics , Osteoblasts/cytology , Parathyroid Hormone/genetics , Parathyroid Hormone/metabolism , Phosphorylation/drug effects , Phosphorylation/physiology , Protein Kinase C/antagonists & inhibitors , Protein Kinase C/genetics , Protein Kinase C/metabolism , Protein Kinase Inhibitors , Serine/genetics , Serine/metabolism , Trans-Activators/geneticsABSTRACT
OBJECTIVE: To determine the role of serine residues at position 63-84 of CITED1 in the nuclear translocation of CITED1 and osteoblast differentiation. METHODS: We engineered all the 9 phosphorylated serine residues of CITED1 with a serine-to-alanine mutation at position 63-84. MC3T3E1 cells transfected with pCDNA3-CFP-CITED1 63-84 (9S>A), pCDNA3-CFP-CITED1, and vehicle plasmid were examined with confocal laser scanning microscopy before and after treatment with 100 nmol/L parathyroid hormone [PTH(1-34)] to observe the changes in the intracellular localization of CITED1. The transfected cells were induced for osteoblastic differentiation with mineralized solution in the absence or presence of 10 nmol/L PTH(1-34), and the changes in ALP activity and Ca(2+) concentration were measured; RT-PCR was used to detect the changes in ALP2, RUNX2, and OC gene expressions after the treatments. RESULTS: s PTH(1-34) promoted the nuclear translocation of CITED1 in MC3T3-E1 cells. The (63-84) 9S>A mutation of CITED1 obviously suppressed its translocation and increased ALP activity and Ca(2+) levels in the cells, which led to enhanced mineralization in the cells with also increased expressions of ALP2, RUNX2, and OC. CONCLUSION: The serine residues at position 63-84 of CITED1 play a vital role in the nuclear translocation of CITED1 and osteoblast differentiation.
Subject(s)
Active Transport, Cell Nucleus , Cell Differentiation , Nuclear Proteins/metabolism , Osteoblasts/cytology , Serine/metabolism , Trans-Activators/metabolism , Animals , Apoptosis Regulatory Proteins , Cell Line , Cell Nucleus , Mice , Mice, Inbred C57BL , Mutation , PlasmidsABSTRACT
OBJECTIVE: To study the effect of signal-selective parathyroid hormone (PTH) analogue peptide on Wnt signaling factors in osteoblasts isolated from neonatal mouse, and provide theoretical basis for the mechanism of PTH's function in bone metabolism. METHODS: Osteoblasts were isolated from calvaria of 2-3-day-old C57BL neonatal mouse and identified by alkaline phosphatase (ALP) staining, and Alizarin red staining. The cells at passage 1 were divided into 4 groups: control group, PTH (1-34) group, G1R19 (1-34) group, and G1R19 (1-28) group. Then the medium was changed to a-MEM supplemented with 1% FBS. After 12 hours, trifluoroacetic acid or three peptides [(10 nmol/L PTH (1-34), 10 nmol/L G1R19 (1-34), and 100 nmol/L G1R19 (1-28)] were added into the culture medium. After 4 hours, the cells were washed gently with cold PBS 3 times before total RNA was isolated. The expressions of Wnt related genes were measured by quantitative real-time PCR. RESULTS: Most of the cells were polygonal and triangular; the cells were positive for ALP staining with blue cytoplasm at 14 days and the Alizarin red staining showed the formation of red mineralized nodules in the special mineralization induction medium at 28 days. The expressions of osteocalcin mRNA and Wnt5b mRNA in PTH (1-34) group, G1R19 (1-34) group, and G1R19 (1-28) group were significantly higher than those in control group (P < 0.05); the expression of Wnt2 mRNA was significantly lower than that in control group (P < 0.05); the expression of beta-catenin mRNA in PTH (1-34) group was significantly higher than that in control group (P < 0.05); the expression of Wnt7b mRNA in PTH (1-34) group and G1R19 (1-34) group was higher than that in control group, and the G1R19 (1-34) group was higher than PTH (1-34) group and G1R19 (1-28) group (P < 0.05). CONCLUSION: In the Wnt-related factors, PTH (1-34) and G1R19 (1-34) affect mainly canonical Wnt signal factors, but the G1R19 (1-28) chiefly acts on non-canonical Wnt signal factors.
Subject(s)
Osteoblasts/metabolism , Osteogenesis/drug effects , Parathyroid Hormone/analogs & derivatives , Parathyroid Hormone/pharmacology , Wnt Proteins/metabolism , Animals , Cells, Cultured , Female , Male , Mice , Mice, Inbred C57BL , Osteoblasts/cytology , Osteoblasts/drug effects , Osteocalcin/genetics , Osteocalcin/metabolism , Parathyroid Hormone-Related Protein/pharmacology , RNA, Messenger/genetics , RNA, Messenger/metabolism , Real-Time Polymerase Chain Reaction , Signal Transduction/physiology , Skull/cytology , Wnt Proteins/genetics , beta Catenin/genetics , beta Catenin/metabolismABSTRACT
OBJECTIVE: To establish a sensitive and direct method for detecting the activation of protein kinase C (PKC) using fluorescence resonance energy transfer (FRET) technique. METHODS: HEK293 cells were transfected with C kinase activity reporter (CKAR) plasmid or/and parathyroid receptor 1 plasmid , and after incubation for 72 h, the fluorescence resonance energy transfer was measured with or without parathyroid or TPA stimulation. RESULTS: TPA reduced the efficiency of FRET and increased the emission ratio of CFP/YFP (C/Y) in HEK293 cells transfected with CKAR. PTH(1-34) could increase the emission ratio of C/Y in HEK293 cells co-transfected with CKAR and PTHR1 but not in cells transfected with CKAR. CONCLUSION: FRET analysis using CKAR can be utilized to detect the activation of PKC, which provides a useful means for studying the signaling pathways associated with PKC.
