ABSTRACT
Bone morphogenetic proteins (BMPs) induce osteoblastic differentiation in myogenic cells via the phosphorylation of Smads. Two types of Smad phosphatases--small C-terminal domain phosphatase 1 (SCP1) and protein phosphatase magnesium-dependent 1A--have been shown to inhibit BMP activity. Here, we report that SCP1 inhibits the osteoblastic differentiation induced by BMP-4, a constitutively active BMP receptor, and a constitutively active form of Smad1. The phosphatase activity of SCP1 was required for this suppression, and the knockdown of SCP1 in myoblasts stimulated the osteoblastic differentiation induced by BMP signaling. In contrast to protein phosphatase magnesium-dependent 1A, SCP1 did not reduce the protein levels of Smad1 and failed to suppress expression of the Id1, Id2, and Id3 genes. Runx2-induced osteoblastic differentiation was suppressed by SCP1 without affecting the transcriptional activity or phosphorylation levels of Runx2. Taken together, these findings suggest that SCP1 may inhibit the osteoblastic differentiation induced by the BMP-Smad axis via Runx2 by suppressing downstream effector(s).
Subject(s)
Bone Morphogenetic Protein 4/pharmacology , Cell Differentiation/drug effects , Osteoblasts/cytology , Osteoblasts/drug effects , Phosphoprotein Phosphatases/metabolism , Smad1 Protein/metabolism , Animals , Blotting, Western , Cells, Cultured , Core Binding Factor Alpha 1 Subunit/genetics , Core Binding Factor Alpha 1 Subunit/metabolism , Humans , Immunohistochemistry , Immunoprecipitation , Mice , Myoblasts/cytology , Myoblasts/drug effects , Phosphoprotein Phosphatases/genetics , Phosphorylation/drug effects , RNA Interference , Reverse Transcriptase Polymerase Chain Reaction , Smad1 Protein/geneticsABSTRACT
We have developed an in vitro model for studying vascular injury. After 7-10 days in a three-dimensional collagen gel culture, capillary-like tubes were formed in the collagen gels. We injured these capillary-like tubes with a laser microdissection system or a scrape method with razors and then examined the collagen gel culture by phase contrast and electron microscopy. After laser injury, profuse necrotic cells were observed around the injured capillary-like tubes and within the tubular lumen compared to the razor injury. We then isolated total RNA from these cultures and prepared cDNA for investigations by quantitative real-time reverse transcription polymerase chain reaction (RT-PCR). Quantitative real time RT-PCR revealed the up-regulation of transcription factor early growth response-1 (Egr-1) after both laser and razor injury, accompanied by the up-regulation of fibroblast growth factor-2 (FGF-2), a proangiogenic factor downstream of Egr-1. The effective laser energy is concentrated on the minute focal spot only. These methods provide a useful in vitro model for studying vascular injury.
Subject(s)
Lasers , Microdissection , Models, Animal , Neovascularization, Physiologic/physiology , Vascular Diseases/pathology , Animals , Early Growth Response Protein 1/biosynthesis , Fibroblast Growth Factor 2/biosynthesis , Male , Mice , Microscopy, Electron , Reverse Transcriptase Polymerase Chain Reaction , Up-RegulationABSTRACT
PURPOSE: This study examined the effects of secretory leukocyte protease inhibitor (SLPI), a protease inhibitor in tears, in allergic conjunctivitis. METHODS: Conjunctiva of male Hartley guinea pigs sensitized with ovalbumin were treated with SLPI or the vehicle 10 min before antigen challenge or simultaneously. The animals were sacrificed after antigen challenges of 0-24 h duration, and the inhibition of eosinophil conjunctival migration and degranulation by SLPI was analyzed histochemically. The effects of SLPI on mast cell chymase and tryptase were also examined. RESULTS: Treatment of sensitized guinea pigs with SLPI suppressed the conjunctival recruitment and degranulation of eosinophils after antigen challenge for 6 h, inhibiting the development of allergic conjunctivitis. The effects of SLPI were observed at concentrations > or =0.1 microM, with a peak at 5 microM. SLPI inhibited chymase in a dose-dependent manner, but had no effect on tryptase. CONCLUSION: The topical SLPI application may be therapeutic in allergic conjunctivitis.
Subject(s)
Conjunctivitis, Allergic/physiopathology , Eosinophils/drug effects , Eosinophils/physiology , Proteins/administration & dosage , Serine Proteinase Inhibitors/administration & dosage , Administration, Topical , Animals , Cell Degranulation , Cell Movement/drug effects , Chymases , Conjunctivitis, Allergic/pathology , Guinea Pigs , Humans , Male , Mast Cells/drug effects , Mast Cells/enzymology , Osmolar Concentration , Proteinase Inhibitory Proteins, Secretory , Secretory Leukocyte Peptidase Inhibitor , Serine Endopeptidases/metabolism , TryptasesABSTRACT
Small pieces of mouse aorta were cultured in collagen gels, and the formation of capillary-like tubes from the aortic explant was observed under phase-contrast and transmission electron microscopes. Migration of fibroblastic cells from the aortic explant occurred in 2 days. After about 10 days of culture, capillary-like tubes from the aortic explant were formed in the collagen gels. An immunohistochemical study on the collagen gel culture revealed the expression of fibroblast growth factor 2 (FGF-2) near the aortic explant at the active initial stage, with random migration of fibroblastic cells expressing FGF-9. mRNA was isolated from these cultures, and reverse transcription-polymerase chain reaction (RT-PCR) of the cultures revealed the expressions of both FGF-2 and FGF-9. Based on our results, we propose that FGF-9 is also related to angiogenic events.
Subject(s)
Cell Culture Techniques/methods , Fibroblast Growth Factor 2/metabolism , Fibroblast Growth Factors/metabolism , Neovascularization, Physiologic/physiology , Animals , Aorta, Thoracic/cytology , Aorta, Thoracic/metabolism , Capillaries/ultrastructure , Cell Movement , Cells, Cultured , Collagen , Culture Media , Fibroblast Growth Factor 2/genetics , Fibroblast Growth Factor 9 , Fibroblast Growth Factors/genetics , Fibroblasts/cytology , Fibroblasts/metabolism , Gels , Immunoenzyme Techniques , Male , Mice , Mice, Inbred ICR , Microscopy, Electron , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
BACKGROUND: We developed a method for reorganizing the mouse small intestine. In the present study, we investigated whether the reorganized small intestine was morphologically and histochemically differentiated. We also evaluated the reorganized small intestine as an in vitro wound healing model. METHODS: Fetal mouse small intestines were dispersed into single cells, which were then cultured to a high density. Newly formed small intestine-like organs on a membrane filter were observed by light and electron microscopy. Alkaline phosphatase (ALPase) activity of the epithelium was analyzed. To evaluate the reorganized small intestine as an in vitro wound healing model, a scalpel was used to cut the reorganized intestine on a membrane, and the healing process was morphologically and immunohistochemically examined. RESULTS: After 6 days in culture, the surface was almost completely coveed with epithelial cells, and villus-like structures were observed. These epithelial cells formed microvilli, and in parallel with this development, ALPase activity of the microvilli increased (from day 4). Twenty-four hours after the cutting, the wound surface was almost completely covered with undifferentiated epithelial cells. The number of acetylated low-density lipoprotein labeled with 1,1,dioctadecyll,3,3,3,3, tetramethyl-indocarbocyanine perchlorate (DiI-Ac-LDL)-positive macrophages increased after cutting. Platelet-derived growth factor (PDGF)-, basic fibroblast growth factor (bFGF)-, matrix metalloproteinase-1 (MMP-1)-positive cells were detected by immunohistochemical staining. CONCLUSIONS: The reorganized small intestine had a morphologically and histochemically differentiated organoid structure, and was useful as an in vitro model for investigating the process of wound healing.
Subject(s)
Intestine, Small/metabolism , Models, Animal , Wound Healing , Animals , Cells, Cultured , Epithelial Cells/metabolism , Fibroblast Growth Factor 2/metabolism , Immunohistochemistry , Intestine, Small/cytology , Intestine, Small/embryology , Matrix Metalloproteinases/metabolism , Membranes, Artificial , Mice , Mice, Inbred ICR , Platelet-Derived Growth Factor/metabolismABSTRACT
The anti-angiogenic effects of thalidomide were examined in mouse aortae grown in a three-dimensional collagen gel-culture. In our in vitro model, (+/-)-thalidomide and (-)-thalidomide exhibited no anti-angiogenic effects. On the other hand, when the culture was treated with thalidomide plus cytochrome P-450, both types of thalidomides significantly inhibited angiogenesis. Co-administration of 100 microg/ml thalidomide plus 200 microg/ml cytochrome P-450 inhibited angiogenesis more strongly than thalidomide plus cytochrome P-450 at other concentrations (10 microg/ml + 200 microg/ml and 100 microg/ml + 20 microg/ml). To study the relation between the anti-angiogenic effect and TNF-alpha, we also evaluated the concentration of TNF-alpha in the culture medium. We found that the concentration of TNF-alpha was correlated to the strength of the anti-angiogenic effect. The inhibition of angiogenesis by thalidomide and cytochrome P-450 takes place through a suppression of TNF-alpha and involves the metabolism of the thalidomide.
