Biomedical subjects

Glen H Nuckolls

Publications and source records attributed to Glen H Nuckolls.

5 recordsLinked to original sources

PubMed2004

Regulation of MMP-13 expression by RUNX2 and FGF2 in osteoarthritic cartilage.

OBJECTIVE: To understand the molecular mechanisms that lead to increased MMP-13 expression and cartilage degeneration during the progression of osteoarthritis (OA), we have investigated the expression of the transcription factor RUNX2 in OA cartilage and the regulation of MMP-13 expression by RUNX2 and FGF2 in articular chondrocytes. DESIGN: RUNX2 and MMP-13 expression in human OA and control cartilage was analyzed by immunohistochemistry. The effects of RUNX2 over-expression, with or without FGF2 treatment, on MMP-13 promoter activity and enzyme accumulation were measured in articular chondrocytes. Inhibitors of MEK/ERK were assayed for their ability to block FGF2 and RUNX2 up-regulation of the MMP-13 promoter. We analyzed RUNX2 phosphorylation in response to FGF2. RESULTS: Fibrillated OA cartilage exhibited increased RUNX2 immunoreactivity when compared to control cartilage. RUNX2 co-localized with MMP-13 in clusters of chondrocytes in fibrillated OA cartilage. RUNX2 over-expression in cultured chondrocytes increased their responsiveness to FGF2 treatment, which led to increased MMP-13 expression. Inhibitors of MEK/ERK signaling blocked up-regulation of the MMP-13 promoter by RUNX2 and FGF2, and also blocked the activation of RUNX2 by FGF2. FGF2 treatment of articular chondrocytes increased RUNX2 phosphorylation approximately 2-fold. CONCLUSIONS: Increased expression of RUNX2 in OA cartilage may contribute to increased expression of MMP-13. FGF2, which is present in OA synovial fluid, activated RUNX2 via the MEK/ERK pathway and increased MMP-13 expression. However, it is unlikely that RUNX2 is a substrate of ERK1/2. RUNX2 expression and activation may be a significant step in the progression of OA by promoting changes in gene expression and chondrocyte differentiation.

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PubMed2003

Smad signaling in mesenchymal and chondroprogenitor cells.

BACKGROUND: Bone morphogenetic proteins (BMPs) are pleiotropic differentiation factors that regulate cell fate determination by orchestrating the activities of downstream signal transducers. Although BMP ligands can elicit signal transduction from heterodimeric combinations of several type-I and type-II receptors, cytoplasmic transducers of the BMP signal include only three known BMP-specific regulatory Smad proteins: Smad1, 5, and 8. In order to determine the combination of signals that regulate chondrogenesis by BMPs, we analyzed the functions of BMP Smad subtypes. METHODS: Multipotential mesenchymal C3H10T1/2 cells and monopotential chondroprogenitor MC615 cells were placed in micromass culture in the presence or absence of BMP4. Chondrogenic differentiation was assayed by measuring Sox9 and type-II collagen gene expression and by alcian blue staining. Transactivation of type-II collagen by regulatory Smads singly, or in combination with Smad4, which partners with regulatory Smads, was assayed by luciferase activity. RESULTS: In the absence of BMP4, mesenchymal cells did not exhibit chondrogenic differentiation, whereas chondroprogenitor cells showed increased cartilage marker expression. In the presence of BMP4, the rate and extent of chondrogenesis increased in a dose-dependent manner for both cell types. We further determined that Smad1 or Smad5, but not Smad8, synergized with Smad4 in the transactivation of the type-II collagen promoter in chondroprogenitor cells. In contrast, Smad8 and Smad4 presented modest synergy in mesenchymal cells. CONCLUSIONS: Taken together, our data suggest that uncommitted mesenchymal cells do not have the cellular competence to respond to the rate-limiting chondroinductive factor BMP. However, in chondroprogenitor cells, BMP stimulates differentiation through mechanisms mediated by Smad1 or Smad5 in combination with Smad4.

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PubMed2003

BMP4 promotes chondrocyte proliferation and hypertrophy in the endochondral cranial base.

Defects in the growth and development of the endochondral bones that comprise the cranial base contribute to several craniofacial dysmorphic syndromes. Since Bone Morphogenetic Protein (BMP) signaling regulates chondrocyte differentiation and endochondral ossification in developing long bones, we have tested the hypothesis that BMP signaling also participates in regulating development of the cranial base. During in vivo developmental progression of the cranial base in mice, a burst of skeletal growth and chondrocyte maturation was identified in the perinatal period. Using a novel serum-free organ culture system, cranial base structures were cultured as explants in the presence of BMP4 or noggin, and analyzed for morphological and molecular changes. Growth of perinatal cranial base explants was inhibited by treatment with noggin, a BMP inhibitor. Exogenous BMP4 promoted cartilage growth, matrix deposition and chondrocyte proliferation in a dose dependent manner. Correspondingly, expression level of the cartilage markers Sox9 and collagen type II were also increased. Alkaline phosphatase and collagen type X expression were up-regulated and expressed in ectopic hypertrophic chondrocytes after treatment of the cultures with 100 ng/ml BMP4 for seven days. This increase in chondrocyte hypertrophy was accompanied by increased indian hedgehog (Ihh) and parathyroid hormone/parathyroid hormone related peptide (PTH/PTHrP) receptor (PPR) expression, but not increased PTHrP expression. We conclude that endogenous BMPs are required to maintain cartilage growth, and exogenous BMP4 can enhance cartilage maturation and induce ectopic chondrocyte hypertrophy in the cranial base. Therefore, appropriate levels of BMP signaling are important for normal cranial base development.

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PubMed2002

The core-binding factor beta subunit is required for bone formation and hematopoietic maturation.

Core-binding factor beta (Cbfbeta) is the common non-DNA-binding subunit of the Cbf family of heterodimeric transcription factors. Mice deficient in Cbfbeta have a severe block in fetal liver hematopoiesis at the stage of hematopoietic stem cell (HSC) emergence. Here we show that by providing Cbfbeta function in endothelial cells and hematopoietic progenitors we can rescue fetal liver hematopoiesis in Cbfbeta-deficient embryos. The rescued mice die at birth, however, with severe defects in skeletal development, though intramembranous ossification occurs to some extent. Fetal liver hematopoiesis is restored at embryonic day (E) 12.5, but by E17.5 significant impairments in lymphopoiesis and myelopoiesis are observed. Thus, we conclude that the Cbfbeta subunit is required for HSC emergence, bone formation and normal differentiation of lymphoid and myeloid lineage cells.

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PubMed2002

Cbfbeta interacts with Runx2 and has a critical role in bone development.

Runx2 (runt-related transcription factor 2, also known as Cbfa1, Osf2 and AML3) is essential for bone development in mice, and mutations in RUNX2 are found in 65-80% of individuals with cleidocranial dysplasia. Although all Runx family members can interact with Cbfbeta (core-binding factor b, encoded by Cbfb), a role for Cbfbeta in bone development has not been demonstrated owing to lethality in Cbfb(-/-) mouse embryos at 12.5 days post coitum (d.p.c.) from hemorrhages and lack of definitive hematopoiesis. Using a 'knock-in' strategy, we generated mouse embryonic stem (ES) cells that express Cbfb fused in-frame to a cDNA encoding green fluorescent protein (GFP). Cbfb(+/GFP) mice had normal life spans and appeared normal, but Cbfb(GFP/GFP) pups died within the first day after birth. The Cbfb(GFP/GFP) mice exhibited a delay in endochondral and intramembranous ossification as well as in chondrocyte differentiation, similar to but less severe than delays observed in Runx2(-/-) mice. We demonstrate that Cbfbeta is expressed in developing bone and forms a functional interaction with Runx2, and that Cbfb(GFP) is a hypomorphic allele. The fusion allele maintains sufficient function in hematopoietic cells to bypass the early embryonic lethality, and identifies a new role for Cbfb in bone development. Our findings raise the possibility that mutations in CBFB may be responsible for some cases of cleidocranial dysplasia that are not linked to mutations in RUNX2.

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