miR-199a, a bone morphogenic protein 2-responsive MicroRNA, regulates chondrogenesis via direct targeting to Smad1.
"MicroRNAs (miRNA) are short non-coding RNA molecules that regulate a variety of biological processes. The role of miRNAs in BMP2-mediated biological processes is of considerable interest. A comparative miRNA array led to the isolation of several BMP2-responsive miRNAs. Among them, miR-199a(*) is of particular interest, because it was reported to be specifically expressed in the skeletal system. Here we demonstrate that miR-199a(*) is an early responsive target of BMP2: its level was dramatically reduced at 5 h, quickly increased at 24 h and remained higher thereafter in the course of BMP2-triggered chondrogenesis of a micromass culture of pluripotent C3H10T1/2 stem cells. miR-199a(*) significantly inhibited early chondrogenesis, as revealed by the reduced expression of early marker genes for chondrogenesis such as cartilage oligomeric matrix protein (COMP), type II collagen, and Sox9, whereas anti-miR-199a(*) increased the expression of these chondrogenic marker genes. A computer-based prediction algorithm led to the identification of Smad1, a well established downstream molecule of BMP-2 signaling, as a putative target of miR-199a(*). The pattern of Smad1 mRNA expression exhibited the mirror opposite of miR-199a(*) expression following BMP-2 induction. Furthermore, miR-199a(*) demonstrated remarkable inhibition of both endogenous Smad1 as well as a reporter construct bearing the 3-untranslated region of Smad1 mRNA. In addition, mutation of miR-199a(*) binding sites in the 3'-untranslated region of Smad1 mRNA abolished miR-199a(*)-mediated repression of reporter gene activity. Mechanism studies revealed that miR-199a(*) inhibits Smad1/Smad4-mediated transactivation of target genes, and that overexpression of Smad1 completely corrects miR-199a(*)-mediated repression of early chondrogenesis. Taken together, miR-199a(*) is the first BMP2 responsive microRNA found to adversely regulate early chondrocyte differentiation via direct targeting of the Smad1 transcription factor."
"Stimulation of C3H10T1/2 cells with BMP-2 resulted in the increased expression of Smad1"
Smad1 may be a pro-chondrogenic target of LSJL.
This study gives conflicting results:
The bone morphogenetic protein 2 signaling mediator Smad1 participates predominantly in osteogenic and not in chondrogenic differentiation in mesenchymal progenitors C3H10T1/2.
"The role of the bone morphogenetic protein (BMP)-signaling mediator Smad1 in osteogenic or chondrogenic differentiation was investigated in murine parental mesenchymal progenitors C3H10T1/2 and its derivatives constitutively expressing BMP-2 (C3H10T1/2-BMP-2) and, therefore, undergo BMP-mediated osteogenic/ chondrogenic development. The functions of the three Smad1 domains, that is, the N-terminal (MH1) domain, the C-terminal (MH2) domain, and the midregional proline-rich linker domain, were documented and compared with full-length Smadl. We showed that expression of the MH2 domain in parental C3H10T1/2 cells was sufficient to initiate osteogenic differentiation. Interestingly, MH1 was sufficient to initiate transcription of osteogenic marker genes like the osteocalcin or parathyroid hormone/parathyroid hormone-related protein (PTH/PTHrP) receptor. However, MH1 interfered with the histologically distinct formation of osteoblast-like cells. A dominant-negative effect on MH2-mediated osteogenic development in C3H10T1/2 cells was observed by the dose-dependent trans-expression of the midregional linker domain. Importantly, in contrast to osteogenic differentiation, Smad1 and its domains do not mimic or interfere with BMP-2-dependent chondrogenic development as monitored by the inability of MH2 to give rise to histologically distinct chondrocytes in parental C3H10T1/2 cells and by the inefficiency of the MH1 or linker domain to interfere with BMP-2-mediated chondrogenic differentiation."
Although the other study merely suggested that Smad1 reduces miR-199 inhibition of chondrogenesis and doesn't necessarily induce it itself.
"The receptor-regulated Smads (R-Smads) Smad1, Smad5, and Smad8 are directly phosphorylated and activated by a BMP type I receptor (IA, IB, and ALK2, which is also the activin IA receptor [ActRIA])" LSJL does not affect Activin I but rather affects Activin II. LSJL also affects BMP receptor II rather than I.
"Smad2 and Smad3 are phosphorylated and activated by activin or TGF-β type I receptors (ALK4/ActRIB and ALK5/TβRI, respectively)"<-LSJL activates TGF-Beta type I receptors.
Convergence of the BMP and EGF signaling pathways on Smad1 in the regulation of chondrogenesis., also provides conflicting data. It indicates that BMP4 activates chondrogenesis by inducing the translocation of Smad4 into the nucleus and that EGF inhibits chondrogenesis by inhibiting Smad1.
Roles of bone morphogenetic protein type I receptors and Smad proteins in osteoblast and chondroblast differentiation.
"The biological effects of type I serine/threonine kinase receptors and Smad proteins were examined using an adenovirus-based vector system. Constitutively active forms of bone morphogenetic protein (BMP) type I receptors (BMPR-IA and BMPR-IB; BMPR-I group)[LSJL induces BMP type RII] and those of activin receptor-like kinase (ALK)-1 and ALK-2 (ALK-1 group) induced alkaline phosphatase activity in C2C12 cells. Receptor-regulated Smads (R-Smads) that act in the BMP pathways, such as Smad1 and Smad5, also induced the alkaline phosphatase activity in C2C12 cells. BMP-6 dramatically enhanced alkaline phosphatase activity induced by Smad1 or Smad5, probably because of the nuclear translocation of R-Smads triggered by the ligand. Inhibitory Smads, i.e., Smad6 and Smad7, repressed the alkaline phosphatase activity induced by BMP-6 or the type I receptors. Chondrogenic differentiation of ATDC5 cells was induced by the receptors of the BMPR-I group but not by those of the ALK-1 group. However, kinase-inactive forms of the receptors of the ALK-1 and BMPR-I groups blocked chondrogenic differentiation. Although R-Smads failed to induce cartilage nodule formation, inhibitory Smads blocked it. Osteoblast differentiation induced by BMPs is thus mediated mainly via the Smad-signaling pathway, whereas chondrogenic differentiation may be transmitted by Smad-dependent and independent pathways."
"The lack of ability of ALK-1 and ALK-2 to induce differentiation of ATDC5 cells suggests that Smads may not be sufficient for chondrogenic differentiation"
Convergence of the BMP and EGF signaling pathways on Smad1 in the regulation of chondrogenesis., also provides conflicting data. It indicates that BMP4 activates chondrogenesis by inducing the translocation of Smad4 into the nucleus and that EGF inhibits chondrogenesis by inhibiting Smad1.
Roles of bone morphogenetic protein type I receptors and Smad proteins in osteoblast and chondroblast differentiation.
"The biological effects of type I serine/threonine kinase receptors and Smad proteins were examined using an adenovirus-based vector system. Constitutively active forms of bone morphogenetic protein (BMP) type I receptors (BMPR-IA and BMPR-IB; BMPR-I group)[LSJL induces BMP type RII] and those of activin receptor-like kinase (ALK)-1 and ALK-2 (ALK-1 group) induced alkaline phosphatase activity in C2C12 cells. Receptor-regulated Smads (R-Smads) that act in the BMP pathways, such as Smad1 and Smad5, also induced the alkaline phosphatase activity in C2C12 cells. BMP-6 dramatically enhanced alkaline phosphatase activity induced by Smad1 or Smad5, probably because of the nuclear translocation of R-Smads triggered by the ligand. Inhibitory Smads, i.e., Smad6 and Smad7, repressed the alkaline phosphatase activity induced by BMP-6 or the type I receptors. Chondrogenic differentiation of ATDC5 cells was induced by the receptors of the BMPR-I group but not by those of the ALK-1 group. However, kinase-inactive forms of the receptors of the ALK-1 and BMPR-I groups blocked chondrogenic differentiation. Although R-Smads failed to induce cartilage nodule formation, inhibitory Smads blocked it. Osteoblast differentiation induced by BMPs is thus mediated mainly via the Smad-signaling pathway, whereas chondrogenic differentiation may be transmitted by Smad-dependent and independent pathways."
"The lack of ability of ALK-1 and ALK-2 to induce differentiation of ATDC5 cells suggests that Smads may not be sufficient for chondrogenic differentiation"
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