Osteogenesis imperfecta (OI) is a skeletal disorder primarily caused by mutations in the type I collagen genes. However, recent investigations have revealed that mutations in the genes encoding for cartilage-associated protein (CRTAP) or prolyl 3-hydroxylase 1 (P3H1) can cause a severe, recessive form of OI. These reports show minimal 3-hydroxylation of key proline residues in type I collagen as a result of CRTAP or P3H1 deficiency and demonstrate the importance of P3H1 and CRTAP to bone structure and development. P3H1 and CRTAP have previously been shown to form a stable complex with cyclophilin B, and P3H1 was shown to catalyze the 3-hydroxylation of specific proline residues in procollagen I in vitro. Here we describe a mouse model in which the P3H1 gene has been inactivated. Our data demonstrate abnormalities in collagen fibril ultrastructure in tendons from P3H1 null mice by electron microscopy. Differences are also seen in skin architecture, as well as in developing limbs by histology. Additionally bone mass and strength were significantly lower in the P3H1 mice as compared with wild-type littermates. Altogether these investigations demonstrate disturbances of collagen fiber architecture in tissues rich in fibrillar collagen, including bone, tendon, and skin. This model system presents a good opportunity to study the underlying mechanisms of recessive OI and to better understand its effects in humans. Osteogenesis imperfecta (OI)2 is an autosomal dominant genetic disorder and is primarily caused by mutations in the genes encoding for type I collagen (COL1A1 and COL1A2). It is characterized as a heterogeneous group of conditions with varying degrees of severity, including bone fragility, low bone mass, susceptibility to fracture, short stature, bowing of the long bones, and moderate to severe kyphoscoliosis (1-3).Recessive OI cases have been reported more recently and have been shown to be caused by mutations in the cartilage-associated protein (CRTAP), prolyl 3-hydroxylase 1 or leprecan (LEPRE1) gene and cyclophilin B (CypB) (4 -9). The majority of these more recently described patients have been shown to have severe to lethal forms of autosomal recessive OI with some distinctive features (5-6, 10). The 3-hydroxylation of key residues in collagen I from these patients was significantly reduced indicating the importance of P3H1 and CRTAP in collagen stability, secretion, and ultimately in bone development. Additionally, the importance of CRTAP in bone development was demonstrated in CRTAP knock-out mice, which show osteochondrodysplasia characterized by severe osteoporosis and decreased osteoid production (4). The CypB knock-out mouse also shows severe OI (11). P3H1 has been shown to be responsible for the modification of the proline into 3(S)-hydroxyproline in the Xaa position of the Gly-Xaa-Yaa repeating sequence of the alpha 1 chain of type I procollagen (12). It is likely that this enzyme also catalyzes modifications in types II and III collagen as well, although this has not yet been shown (13). P3H1 e...
PAGE 17257:Fig. 2, line 9 should read: F, a projection through a 350 nm thick section of longitudinally sectioned P3H1 null tendon from which the tilt series (supplemental Fig. S1) was collected. ADDITIONS AND CORRECTIONS This paper is available online at www.jbc.orgWe suggest that subscribers photocopy these corrections and insert the photocopies in the original publication at the location of the original article. Authors are urged to introduce these corrections into any reprints they distribute. Secondary (abstract) services are urged to carry notice of these corrections as prominently as they carried the original abstracts.
Hyaluronan (HA) is a major component of the extracellular matrix (ECM) and is synthesized by three HA synthases (HAS). Similarities between the HAS2 knockout mouse and the hdf mutant mouse, which has a mutation in the versican gene, suggest that HA and versican expression may be linked. In this study, the relationship between HA synthesis and levels of versican, fibronectin and several other ECM components in trabecular meshwork cells from the anterior segment of the eye was investigated. HA synthesis was inhibited using 4-methylumbelliferone (4MU), or reduced by RNAi silencing of each individual HAS gene. Quantitative RT-PCR and immunoblotting demonstrated a reduction in mRNA and protein levels of versican and fibronectin. Hyaluronidase treatment also reduced versican and fibronectin levels. These effects could not be reversed by addition of excess glucose or glucosamine or exogenous HA to the culture medium. CD44, tenascin C and fibrillin-1 mRNA levels were reduced by 4MU treatment, but SPARC and CSPG6 mRNA levels were unaffected. Immunostaining of trabecular meshwork tissue after exposure to 4MU showed an altered localization pattern of HA-binding protein, versican and fibronectin. Reduction of versican by RNAi silencing did not affect HA concentration as assessed by ELISA. Together, these data imply that HA concentration affects synthesis of certain ECM components. Since precise regulation of the trabecular meshwork ECM composition and organization is required to maintain the aqueous humor outflow resistance and intraocular pressure homeostasis in the eye, coordinated coupling of HA levels and several of its ECM binding partners should facilitate this process.
Environmental toxicants such as bisphenol-A (BPA) and polychlorinated biphenyls (PCBs) are prevalent in our water supply, soil, and many food products and can profoundly affect the central nervous system. Both BPA and PCBs can disrupt endocrine signaling, which is important for auditory development and function, but the effect of these toxicants on the auditory periphery is not understood. In this study we investigated the effect of PCB-95 and BPA on lateral line development, function, and regeneration in larval zebrafish. The lateral line is a system of mechanosensory hair cells on the exterior of the fish that are homologous to the hair cells located in the mammalian inner ear. We found that PCB-95 had no effect on lateral line development or hair cell survival. BPA also did not affect lateral line development, but instead had a significant effect on both hair cell survival and regeneration. BPA-induced hair cell loss is both dose-and time-dependent, with concentrations of 1 μM or higher killing lateral line hair cells during a 24 hr exposure period. Pharmacologic manipulation experiments suggest that BPA kills hair cells via activation of oxidative stress pathways, similar to prior reports of BPA toxicity in other tissues. We also observed that hair cells killed with neomycin, a known ototoxin, failed to regenerate normally when BPA was present, suggesting that BPA in aquatic environments could impede innate regenerative responses in fishes. Collectively, these data demonstrate that BPA can have detrimental effects on sensory systems, both in aquatic life and perhaps in terrestrial organisms, including humans.
Inner ear hair cell death leads to sensorineural hearing loss and can be a direct consequence of aminoglycoside antibiotic treatment. Aminoglycosides such as gentamicin are effective therapy for serious Gram-negative bacterial infections such as some forms of meningitis, pneumonia, and sepsis. Aminoglycosides enter hair cells through mechanotransduction channels at the apical end of hair bundles and initiate intrinsic cell death cascades, but the precise cell signaling that leads to hair cell death is incompletely understood. Here, we examine the cell death pathways involved in aminoglycoside damage using the zebrafish (Danio rerio). The zebrafish lateral line contains hair cell-bearing organs called neuromasts that are homologous to hair cells of the mammalian inner ear and represents an excellent model to study ototoxicity. Based on previous research demonstrating a role for p53, Bcl2 signaling, autophagy, and proteasomal degradation in aminoglycoside-damaged hair cells, we used the Cytoscape GeneMANIA Database to identify additional proteins that might play a role in neomycin or gentamicin ototoxicity. Our bioinformatics analysis identified the pro-survival proteins phosphoinositide-dependent kinase-1 (PDK1) and X-linked inhibitor of apoptosis protein (Xiap) as potential mediators of gentamicin-induced hair cell damage. Pharmacological inhibition of PDK1 or its downstream mediator protein kinase C facilitated gentamicin toxicity, as did Xiap mutation, suggesting that both PI3K and endogenous Xiap confer protection. Surprisingly, aminoglycoside-induced hair cell death was highly attenuated in wild type Tupfel long-fin (TL fish; the background strain for the Xiap mutant line) compared to wild type ∗AB zebrafish. Pharmacologic manipulation of p53 suggested that the strain difference might result from decreased p53 in TL hair cells, allowing for increased hair cell survival. Overall, our studies identified additional steps in the cell death cascade triggered by aminoglycoside damage, suggesting possible drug targets to combat hearing loss resulting from aminoglycoside exposure.
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