Osteogenic Factor Runx2 Marks a Subset of Leptin Receptor-Positive Cells that Sit Atop the Bone Marrow Stromal Cell Hierarchy

Bone marrow mesenchymal stem and progenitor cells (BM-MSPCs) maintain homeostasis of bone tissue by providing osteoblasts. Although several markers have been identified for labeling of MSPCs, these labeled cells still contain non-BM-MSPC populations. Studies have suggested that MSPCs are observed as leptin receptor (LepR)-positive cells, whereas osteoblasts can be classified as positive for Runx2, a master regulator for osteoblastogenesis. Here, we demonstrate, using Runx2-GFP reporter mice,that the LepR-labeled population contains Runx2-GFPlow sub-population, which possesses higher fibroblastic colony-forming units (CFUs) and mesensphere capacity, criteria for assessing stem cell activity, than the Runx2-GFP− population. In response to parathyroid hormone (PTH), a bone anabolic hormone, LepR+Runx2-GFPlow cells increase Runx2 expression and form multilayered structures near the bone surface. Subsequently, the multilayered cells express Osterix and Type I collagen α, resulting in generation of mature osteoblasts. Therefore, our results indicate that Runx2 is weakly expressed in the LepR+ population without osteoblastic commitment, and the LepR+Runx2-GFPlow stromal cells sit atop the BM stromal hierarchy

Three synonymous genes encode calmodulin in a reptile, the Japanese tortoise, Clemmys japonica

Three distinct calmodulin (CaM)-encoding cDNAs were isolated from a reptile, the Japanese tortoise (Clemmys japonica), based on degenerative primer PCR. Because of synonymous codon usages, the deduced amino acid (aa) sequences were exactly the same in all three genes and identical to the aa sequence of vertebrate CaM. The three cDNAs, referred to as CaM-A, -B, and -C, seemed to belong to the same type as CaMI, CaMII, and CaMIII, respectively, based on their sequence identity with those of the mammalian cDNAs and the glutamate codon biases. Northern blot analysis detected CaM-A and -B as bands corresponding to 1.8 kb, with the most abundant levels in the brain and testis, while CaM-C was detected most abundantly in the brain as bands of 1.4 and 2.0 kb. Our results indicate that, in the tortoise, CaM protein is encoded by at least three non-allelic genes, and that the ‘multigene-one protein' principle of CaM synthesis is applicable to all classes of vertebrates, from fishes to mammals

Involvement of Fibronectin and Matrix Metalloproteinases in Airway Smooth Muscle Cell Migration for the Process of Airway Remodeling

ABSTRACT: Background: Airway remodeling is a repair process occurring after airway injury; its primary histopathological features are subepithelial fibrosis and smooth muscle thickening of the bronchi. These histopathological changes are considered to occur due to bronchial smooth muscle cells (bSMC) that secrete extracellular matrix (ECM) proteins, which work as chemoattractants and influence cell migration. Therefore, we examined the interaction between bSMCs and ECM proteins in vitro for understanding the remodeling process in the bronchi. Methods: bSMCs were cultured to collect a bSMC-conditioned medium. Using the bSMC-conditioned medium thus obtained, we performed a cell migration assay, characterized p integrin expression, and identified ECM proteins and matrix metalloproteinases by western blotting and gelatin zymography, respectively. Results: The response of bSMC migration to bSMC-conditioned medium increased with time in culture, and fibronectin (FIB) was detected as a chemoattractant for bSMCs in bSMC-conditioned medium by western blot analysis and a cell migration assay using anti-FIB antibodies. The involvement of p1 integrin in the migration of bSMCs toward FIB contained in bSMC-conditioned medium was demonstrated by inhibition of cell migration using anti-p1 integrin antibodies. Expression of p1 integrin on bSMCs was confirmed by using a p-integrinmediated cell adhesion array. In addition, metalloproteinases detected in bSMC-conditioned medium by gelatin zymography were suggested to be matrix metalloproteinase-1 and 2 by western blotting and amino acid sequencing. Conclusions: Our results suggest that FIB and matrix metalloproteinases secreted from bSMCs might play major roles in bSMC migration in the process of airway remodeling. KEY WORDS: airway remodeling, cell migration, fibronectin, matrix metalloproteinases, smooth muscle cell