ABSTRACT
It has been suggested that some adult bone marrow cells (BMC) can localize to the lung and develop tissue-specific characteristics including those of pulmonary epithelial cells. Here, we show that the combination of mild airway injury (naphthalene-induced) as a conditioning regimen to direct the site of BMC localization and transtracheal delivery of short-term cultured BMC enhances airway localization and adoption of an epithelial-like phenotype. Confocal analysis of airway and alveolar-localized BMC (fluorescently labeled) with epithelial markers shows expression of the pulmonary epithelial proteins, Clara cell secretory protein, and surfactant protein C. To confirm epithelial gene expression by BMC, we generated transgenic mice expressing green fluorescent protein (GFP) driven by the epithelial-specific cytokeratin-18 promoter and injected BMC from these mice transtracheally into wild-type recipients after naphthalene-induced airway injury. BMC retention in the lung was observed for at least 120 days following cell delivery with increasing GFP transgene expression over time. Some BMC cultured in vitro over time also expressed GFP transgene, suggesting epithelial transdifferentiation of the BMC. The results indicate that targeted delivery of BMC can promote airway regeneration.
Subject(s)
Bone Marrow Cells/cytology , Epithelial Cells/cytology , Regeneration , Respiratory System/cytology , Animals , Biomarkers/metabolism , Cell Differentiation , Cell Fusion , Cells, Cultured , Female , Gene Expression Regulation , Green Fluorescent Proteins/metabolism , Immunophenotyping , Keratin-18/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Organ Specificity , Promoter Regions, Genetic/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Respiratory System/pathology , Trachea/cytology , Transgenes , Uteroglobin/metabolismABSTRACT
CSX/Nkx2.5 transcription factor plays a pivotal role in cardiac development; however, its role in development and differentiation of other organs has not been investigated. In this study, we used C2C12 myoblasts and human fetal primary myoblasts to investigate the function of Nkx2.5 in skeletal myogenesis. The expression levels of Nkx2.5 decreased as C2C12 myoblasts elongated and fused to form myotubes. The expression of human NKX2.5 in C2C12 myoblasts inhibited myocyte differentiation and myotube formation, and up-regulated Gata4 and Tbx5 expression. The expression of NKX2.5 in terminally differentiated C2C12 myotubes resulted in a change in morphology and breakdown into smaller myotubes. Furthermore, overexpression of NKX2.5 in C2C12 cells and primary cultures of human fetal myoblasts led to differentiation of myoblasts into neuron-like cells and expression of neuronal markers. This study sheds light on the previously unknown non-cardiac functions of Nkx2.5 transcription factor.
Subject(s)
Homeodomain Proteins/physiology , Muscle, Skeletal/cytology , Muscles/embryology , Transcription Factors/physiology , Adenoviridae/genetics , Animals , Blotting, Western , Bromodeoxyuridine/pharmacology , Cell Cycle , Cell Differentiation , Cell Line , Cell Nucleus/metabolism , Cells, Cultured , Culture Media/pharmacology , DNA-Binding Proteins/biosynthesis , GATA4 Transcription Factor , Green Fluorescent Proteins/metabolism , Homeobox Protein Nkx-2.5 , Homeodomain Proteins/metabolism , Humans , Immunoprecipitation , Mice , Microscopy, Fluorescence , Muscles/cytology , Muscles/metabolism , Myogenin/metabolism , Neurons/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction , T-Box Domain Proteins/biosynthesis , Transcription Factors/biosynthesis , Transcription Factors/metabolism , Up-RegulationABSTRACT
We have identified 16 different mutations of the low-density lipoprotein receptor (LDLR) gene in 25 unrelated Korean patients with heterozygous familial hypercholesterolemia (FH), including five novel mutations, C83Y, 661del17, 1705insCTAG, C675X, and 941-1G>A. The 1705insCTAG mutation in which the four 3 cent -terminal nucleotides of exon 11 are duplicated was found to prevent splicing of exon 11 and would therefore generate a truncated polypeptide. The in-frame 36-bp deletion (1591del36) in exon 11, which had been reported only in one Korean FH patient, was also found. We showed that this change affects transport of the LDL receptor from the endoplasmic reticulum to the cell surface. In addition, we found 8 mutations (-136C>T, E119K, E207K, E207X, F382L, R574Q, 1846-1G>A, and P664L) that had been described in other ethnic groups but not in Koreans, and 2 mutations (R94H and D200N) that had been described in Koreans as well as other ethnic groups. 5 mutations (1591del36, E119K, E207X, E207K, and P664L) were found more than once in the Korean FH samples. Identification of the novel and recurring LDLR mutations in Korean FH patients should facilitate prenatal and early diagnosis in families at high risk of FH.
