Functional biology of the Steel syndrome founder allele and evidence for clan genomics derivation of COL27A1 pathogenic alleles worldwide.

Previously we reported the identification of a homozygous COL27A1 (c.2089G>C; p.Gly697Arg) missense variant and proposed it as a founder allele in Puerto Rico segregating with Steel syndrome (STLS, MIM #615155); a rare osteochondrodysplasia characterized by short stature, congenital bilateral hip dysplasia, carpal coalitions, and scoliosis. We now report segregation of this variant in five probands from the initial clinical report defining the syndrome and an additional family of Puerto Rican descent with multiple affected adult individuals. We modeled the orthologous variant in murine Col27a1 and found it recapitulates some of the major Steel syndrome associated skeletal features including reduced body length, scoliosis, and a more rounded skull shape. Characterization of the in vivo murine model shows abnormal collagen deposition in the extracellular matrix and disorganization of the proliferative zone of the growth plate. We report additional COL27A1 pathogenic variant alleles identified in unrelated consanguineous Turkish kindreds suggesting Clan Genomics and identity-by-descent homozygosity contributing to disease in this population. The hypothesis that carrier states for this autosomal recessive osteochondrodysplasia may contribute to common complex traits is further explored in a large clinical population cohort. Our findings augment our understanding of COL27A1 biology and its role in skeletal development; and expand the functional allelic architecture in this gene underlying both rare and common disease phenotypes.

Role of uL3 in Multidrug Resistance in p53-Mutated Lung Cancer Cells.

Cancer is one of the most common causes of death among adults. Chemotherapy is crucial in determining patient survival and quality of life. However, the development of multidrug resistance (MDR) continues to pose a significant challenge in the management of cancer. In this study, we analyzed the role of human ribosomal protein uL3 (formerly rpL3) in multidrug resistance. Our studies revealed that uL3 is a key determinant of multidrug resistance in p53-mutated lung cancer cells by controlling the cell redox status. We established and characterized a multidrug resistant Calu-6 cell line. We found that uL3 down-regulation correlates positively with multidrug resistance. Restoration of the uL3 protein level re-sensitized the resistant cells to the drug by regulating the reactive oxygen species (ROS) levels, glutathione content, glutamate release, and cystine uptake. Chromatin immunoprecipitation experiments and luciferase assays demonstrated that uL3 coordinated the expression of stress-response genes acting as transcriptional repressors of solute carrier family 7 member 11 (xCT) and glutathione S-transferase α1 (GST-α1), independently of Nuclear factor erythroid 2-related factor 2 (Nrf2). Altogether our results describe a new function of uL3 as a regulator of oxidative stress response genes and advance our understanding of the molecular mechanisms underlying multidrug resistance in cancers.

Fibrillin-1 microfibrils influence adult bone marrow hematopoiesis.

We have recently demonstrated that fibrillin-1 assemblies regulate the fate of skeletal stem cells (aka, mesenchymal stem cells [MSCs]) by modulating TGFß activity within the microenvironment of adult bone marrow niches. Since MSCs can also influence hematopoietic stem cell (HSC) activities, here we investigated adult hematopoiesis in mice with Cre-mediated inactivation of the fibrillin-1 (Fbn1) gene in the mesenchyme of the forming limbs (Fbn1(Prx1-/-) mice). Analyses of 3-month-old Fbn1(Prx1-/-) mice revealed a statistically significant increase of circulating red blood cells, which a differentiation assay correlated with augmented erythropoiesis. This finding, together with evidence of fibrillin-1 deposition in erythroblastic niches, supported the notion that this extracellular matrix protein normally restricts differentiation of erythroid progenitors. Whereas flow cytometry measurements identified a decreased HSC frequency in mutant relative to wild type mice, no appreciable differences were noted with regard to the relative abundance and differentiation potential of myeloid progenitor cells. Together these findings implied that fibrillin-1 normally promotes HSC expansion but does not influence cell lineage commitment. Since local TGFß hyperactivity has been associated with abnormal osteogenesis in Fbn1(Prx1-/-) mice, 1-month-old mutant and wild type animals were systemically treated for 8weeks with either a pan-TGF-ß-neutralizing antibody or an antibody of the same IgG1 isotype. The distinct outcomes of these pharmacological interventions strongly suggest that fibrillin-1 differentially modulates TGFß activity in HSC vs. erythroid niches.

Fibrillin microfibrils in bone physiology.

The severe skeletal abnormalities associated with Marfan syndrome (MFS) and congenital contractural arachnodactyly (CCA) underscore the notion that fibrillin assemblies (microfibrils and elastic fibers) play a critical role in bone formation and function in spite of representing a low abundance component of skeletal matrices. Studies of MFS and CCA mice have correlated the skeletal phenotypes of these mutant animals with distinct pathophysiological mechanisms that reflect the contextual contribution of fibrillin-1 and -2 scaffolds to TGFß and BMP signaling during bone patterning, growth and metabolism. Illustrative examples include the unique role of fibrillin-2 in regulating BMP-dependent limb patterning and the distinct impact of the two fibrillin proteins on the commitment and differentiation of marrow mesenchymal stem cells. Collectively, these findings have important implication for our understanding of the pathophysiological mechanisms that drive age- and injury-related processes of bone degeneration.

Fibrillin-1 Regulates Skeletal Stem Cell Differentiation by Modulating TGFß Activity Within the Marrow Niche.

A full understanding of the microenvironmental factors that control the activities of skeletal stem cells (also known as mesenchymal stem cells [MSCs]) in the adult bone marrow holds great promise for developing new therapeutic strategies to mitigate age-related diseases of bone and cartilage degeneration. Bone loss is an understudied manifestation of Marfan syndrome, a multisystem disease associated with mutations in the extracellular matrix protein and TGFß modulator fibrillin-1. Here we demonstrate that progressive loss of cancellous bone in mice with limbs deficient for fibrillin-1 (Fbn1(Prx1-/-) mice) is accounted for by premature depletion of MSCs and osteoprogenitor cells combined with constitutively enhanced bone resorption. Longitudinal analyses of Fbn1(Prx1-/-) mice showed incremental bone loss and trabecular microarchitecture degeneration accompanied by a progressive decrease in the number and clonogenic potential of MSCs. Significant paucity of marrow fat cells in the long bones of Fbn1(Prx1-/-) mice, together with reduced adipogenic potential of marrow stromal cell cultures, indicated an additional defect in MSC differentiation. This postulate was corroborated by showing that an Fbn1-silenced osteoprogenitor cell line cultured in the presence of insulin yielded fewer than normal adipocytes and exhibited relatively lower PPARγ levels. Consonant with fibrillin-1 modulation of TGFß bioavailability, cultures of marrow stromal cells from Fbn1(Prx1-/-) limb bones showed improper overactivation of latent TGFß. In line with this finding, systemic TGFß neutralization improved bone mass and trabecular microarchitecture along with normalizing the number of MSCs, osteoprogenitor cells, and marrow adipocytes. Collectively, our findings show that fibrillin-1 regulates MSC activity by modulating TGFß bioavailability within the microenvironment of marrow niches.

Ha-Ras stabilization mediates pro-fibrotic signals in dermal fibroblasts.

BACKGROUND: Scleroderma (systemic sclerosis; SSc) is a clinically heterogeneous and often lethal acquired disorder of the connective tissue that is characterized by vascular, immune/inflammatory and fibrotic manifestations. Tissue fibrosis is the main cause of morbidity and mortality in SSc and an unmet medical challenge, mostly because of our limited understanding of the molecular factors and signalling events that trigger and sustain disease progression. Recent evidence has correlated skin fibrosis in SSc with stabilization of proto-oncogene Ha-Ras secondary to auto-antibody stimulation of reactive oxygen species production. The goal of the present study was to explore the molecular connection between Ha-Ras stabilization and collagen I production, the main read-out of fibrogenesis, in a primary dermal fibroblast culture system that replicates the early stages of disease progression in SSc. RESULTS: Forced expression of proto-oncogene Ha-Ras in dermal fibroblasts demonstrated the promotion of an immediate collagen I up-regulation, as evidenced by enhanced activity of a collagen I-driven luciferase reporter plasmid and increased accumulation of endogenous collagen I proteins. Moreover, normal levels of Tgfß transcripts and active transforming growth factor-beta (TGFß) implied Ha-Ras stimulation of the canonical Smad2/3 signalling pathway independently of TGFß production or activation. Heightened Smad2/3 signalling was furthermore correlated with greater Smad3 phosphorylation and Smad3 protein accumulation, suggesting that Ha-Ras may target both Smad2/3 activation and turnover. Additional in vitro evidence excluded a contribution of ERK1/2 signalling to improper Smad3 activity and collagen I production in cells that constitutively express Ha-Ras. CONCLUSIONS: Our study shows for the first time that constitutively elevated Ha-Ras protein levels can directly stimulate Smad2/3 signalling and collagen I accumulation independently of TGFß neo-synthesis and activation. This finding therefore implicates the Ha-Ras pathway with the early onset of fibrosis in SSc and implicitly identifies new therapeutic targets in SSc.

Extracellular microfibrils control osteoblast-supported osteoclastogenesis by restricting TGF{beta} stimulation of RANKL production.

Mutations in fibrillin-1 or fibrillin-2, the major structural components of extracellular microfibrils, cause pleiotropic manifestations in Marfan syndrome and congenital contractural arachnodactyly, respectively. We recently found that fibrillin-1 and fibrillin-2 control bone formation by regulating osteoblast differentiation through the differential modulation of endogenous TGFß and bone morphogenetic protein signals. Here, we describe in vivo and ex vivo experiments that implicate the fibrillins as negative regulators of bone resorption. Adult Fbn2(-/-) mice display a greater than normal osteolytic response to locally implanted lipopolysaccharide-coated titanium particles. Although isolated cultures of Fbn2(-/-) preosteoclasts exhibited normal differentiation and activity, these features were substantially augmented when mutant or wild-type preosteoclasts were co-cultured with Fbn2(-/-) but not wild-type osteoblasts. Greater osteoclastogenic potential of Fbn2(-/-) osteoblasts was largely accounted for by up-regulation of the Rankl gene secondary to heightened TGFß activity. This conclusion was based on the findings that blockade of TGFß signaling blunts Rankl up-regulation in Fbn2(-/-) osteoblasts and bones and that systemic TGFß antagonism improves locally induced osteolysis in Fbn2(-/-) mice. Abnormally high Rankl expression secondary to elevated TGFß activity was also noted in cultured osteoblasts from Fbn1(-/-) mice. Collectively our data demonstrated that extracellular microfibrils balance local catabolic and anabolic signals during bone remodeling in addition to implying distinct mechanisms of bone loss in Marfan syndrome and congenital contractural arachnodactyly.

Identification of the Drosophila progenitor of mammalian Krüppel-like factors 6 and 7 and a determinant of fly development.

The Krüppel-like transcription factors (KLFs) represent a family of 15 different zinc finger proteins of the C(2)H(2) type that are involved in vertebrate development and which control cell proliferation, growth and differentiation. Structural-functional considerations have segregated KLF6 and KLF7 into a phylogenetically distinct group. Here we report the identification of Luna, the Drosophila progenitor of the mammalian KLF6/KLF7 group. This conclusion is based on the near sequence identity, as well as the comparable location of the DNA-binding domains and nuclear localization signals of the insect and mammalian proteins. The homology extends to the composition and function of the amino-terminal segment of Luna which, similarly to the mammalian counterparts, stimulates transcription in a reporter gene assay. We also present preliminary in vivo evidence of Luna involvement in embryonic development and cell differentiation. First, luna RNA interference and luna overexpression during early Drosophila embryogenesis leads to developmental arrest at different embryonic stages. Second, targeted perturbation of luna expression in the forming compound eye interferes with terminal cell differentiation, but not cell specification. We therefore propose that Luna is a novel transcriptional determinant of Drosophila development.

The human homologue of the mouse Surf5 gene encodes multiple alternatively spliced transcripts.

Hu-Surf5 is included within the Surfeit locus, a cluster of six genes originally identified in mouse. In the present study, we have cloned and characterized the Hu-Surf5 gene and its mRNA multiple transcripts. Comparison of the most abundant cDNA and genomic sequence shows that the Hu-Surf5 is spread over a region of approximately 7.5 kb and consists of five exons separated by four introns. The nucleotide sequence of the genomic region flanking the 3'-end of the Hu-Surf5 gene revealed the presence of a processed pseudogene of human ribosomal protein L21 followed by Hu-Surf6 gene. Only 110 bp separate the transcription start site of Hu-Surf5 and Hu-Surf3/L7a gene and the transcription direction is divergent. Earlier studies defined the 110 bp region essential for promoter activity of Hu-Surf3/L7a. Here, we show that this region stimulates transcription with a slightly different efficiency in both directions. The bidirectional promoter lacks an identifiable TATA box and is characterized by a CpG island that extends through the first exon into the first intron of both genes. These features are characteristic of housekeeping genes and are consistent with the wide tissue distribution observed for Hu-Surf5 expression. Hu-Surf5 encodes three different transcripts, Surf-5a, Surf-5b, and Surf-5c, which result from alternative splicing. Two protein products, SURF-5A and SURF-5B have been characterized. Production of chimaeras between the full-length SURF-5A or SURF-5B and the green fluorescent protein (GFP) allowed to localize both proteins in the cytoplasm.