Transcriptional regulation of mouse alpha A-crystallin gene in a 148kb Cryaa BAC and its derivates

<p>Abstract</p> <p>Background</p> <p>αA-crystallin is highly expressed in the embryonic, neonatal and adult mouse lens. Previously, we identified two novel distal control regions, DCR1 and DCR3. DCR1 was required for transgenic expression of enhanced green fluorescent protein, EGFP, in lens epithelium, whereas DCR3 was active during "late" stages of lens primary fiber cell differentiation. However, the onset of transgenic EGFP expression was delayed by 12–24 hours, compared to the expression of the endogenous <it>Cryaa </it>gene.</p> <p>Results</p> <p>Here, we used bacterial artificial chromosome (BAC) and standard transgenic approaches to examine temporal and spatial regulation of the mouse <it>Cryaa </it>gene. Two BAC transgenes, with EGFP insertions into the third coding exon of <it>Cryaa </it>gene, were created: the intact α<it>A-crystallin </it>148 kb BAC (αA-BAC) and αA-BAC(ΔDCR3), which lacks approximately 1.0 kb of genomic DNA including DCR3. Expression of EGFP in the majority of both BAC transgenics nearly recapitulated the endogenous expression pattern of the <it>Cryaa </it>gene in lens, but not outside of the lens. The number of cells expressing αA-crystallin in the lens pit was higher compared to the number of cells expressing EGFP. Next, we generated additional lines using a 15 kb fragment of α<it>A-crystallin </it>locus derived from αA-BAC(ΔDCR3), 15 kb <it>Cryaa/EGFP</it>. A 15 kb region of <it>Cryaa/EGFP </it>supported the expression pattern of EGFP also in the lens pit. However, co-localization studies of αA-crystallin and EGFP indicated that the number of cells that showed transgenic expression was higher compared to cells expressing αA-crystallin in the lens pit.</p> <p>Conclusion</p> <p>We conclude that a 148 kb αA-BAC likely contains all of the regulatory regions required for αA-crystallin expression in the lens, but not in retina, spleen and thymus. In addition, while the 15 kb <it>Cryaa/EGFP </it>region also supported the expression of EGFP in the lens pit, expression in regions such as the hindbrain, indicate that additional genomic regions may play modulatory functions in regulating extralenticular αA-crystallin expression. Finally, deletion of DCR3 in either αA-BAC(ΔDCR3) or <it>Cryaa </it>(15 kb) transgenic mice result in EGFP expression patterns that are consistent with DCR's previously established role as a distal enhancer active in "late" primary lens fiber cells.</p

Identification of the HSPB4/TLR2/NF-κB axis in macrophage as a therapeutic target for sterile inflammation of the cornea

Sterile inflammation underlies many diseases of the cornea including serious chemical burns and the common dry eye syndrome. In search for therapeutic targets for corneal inflammation, we defined the kinetics of neutrophil infiltration in a model of sterile injury to the cornea and identified molecular and cellular mechanisms triggering inflammatory responses. Neutrophil infiltration occurred in two phases: a small initial phase (Phase I) that began within 15 min after injury, and a larger second phase (Phase II) that peaked at 24–48 h. Temporal analysis suggested that the neuropeptide secretoneurin initiated Phase I without involvement of resident macrophages. Phase II was initiated by the small heat shock protein HSPB4 that was released from injured keratocytes and that activated resident macrophages via the TLR2/NF-κB pathway. The Phase II inflammation was responsible for vision-threatening opacity and was markedly suppressed by different means of inhibition of the HSPB4/TLR2/NF-κB axis: in mice lacking HSPB4 or TLR2, by antibodies to HSPB4 or by TNF-α stimulated gene/protein 6 that CD44-dependently inhibits the TLR2/NF-κB pathway. Therefore, our data identified the HSPB4/TLR2/NF-κB axis in macrophages as an effective target for therapy of corneal inflammation

DNA2 drives processing and restart of reversed replication forks in human cells

Accurate processing of stalled or damaged DNA replication forks is paramount to genomic integrity and recent work points to replication fork reversal and restart as a central mechanism to ensuring high-fidelity DNA replication. Here, we identify a novel DNA2- and WRN-dependent mechanism of reversed replication fork processing and restart after prolonged genotoxic stress. The human DNA2 nuclease and WRN ATPase activities functionally interact to degrade reversed replication forks with a 5'-to-3' polarity and promote replication restart, thus preventing aberrant processing of unresolved replication intermediates. Unexpectedly, EXO1, MRE11, and CtIP are not involved in the same mechanism of reversed fork processing, whereas human RECQ1 limits DNA2 activity by preventing extensive nascent strand degradation. RAD51 depletion antagonizes this mechanism, presumably by preventing reversed fork formation. These studies define a new mechanism for maintaining genome integrity tightly controlled by specific nucleolytic activities and central homologous recombination factors

Perturbing the Ubiquitin Pathway Reveals How Mitosis Is Hijacked to Denucleate and Regulate Cell Proliferation and Differentiation In Vivo

The eye lens presents a unique opportunity to explore roles for specific molecules in cell proliferation, differentiation and development because cells remain in place throughout life and, like red blood cells and keratinocytes, they go through the most extreme differentiation, including removal of nuclei and cessation of protein synthesis. Ubiquitination controls many critical cellular processes, most of which require specific lysines on ubiquitin (Ub). Of the 7 lysines (K) least is known about effects of modification of K6.We replaced K6 with tryptophan (W) because K6 is the most readily modified K and W is the most structurally similar residue to biotin. The backbone of K6W-Ub is indistinguishable from that of Wt-Ub. K6W-Ub is effectively conjugated and deconjugated but the conjugates are not degraded via the ubiquitin proteasome pathways (UPP). Expression of K6W-ubiquitin in the lens and lens cells results in accumulation of intracellular aggregates and also slows cell proliferation and the differentiation program, including expression of lens specific proteins, differentiation of epithelial cells into fibers, achieving proper fiber cell morphology, and removal of nuclei. The latter is critical for transparency, but the mechanism by which cell nuclei are removed has remained an age old enigma. This was also solved by expressing K6W-Ub. p27(kip), a UPP substrate accumulates in lenses which express K6W-Ub. This precludes phosphorylation of nuclear lamin by the mitotic kinase, a prerequisite for disassembly of the nuclear membrane. Thus the nucleus remains intact and DNAseIIβ neither gains entry to the nucleus nor degrades the DNA. These results could not be obtained using chemical proteasome inhibitors that cannot be directed to specific tissues.K6W-Ub provides a novel, genetic means to study functions of the UPP because it can be targeted to specific cells and tissues. A fully functional UPP is required to execute most stages of lens differentiation, specifically removal of cell nuclei. In the absence of a functional UPP, small aggregate prone, cataractous lenses are formed

Transketolase Haploinsufficiency Reduces Adipose Tissue and Female Fertility in Mice

Transketolase (TKT) is a ubiquitous enzyme used in multiple metabolic pathways. We show here by gene targeting that TKT-null mouse embryos are not viable and that disruption of one TKT allele can cause growth retardation (≈35%) and preferential reduction of adipose tissue (≈77%). Other TKT(+/−) tissues had moderate (≈33%; liver, gonads) or relatively little (≈7 to 18%; eye, kidney, heart, brain) reductions in mass. These mice expressed a normal level of growth hormone and reduced leptin levels. No phenotype was observed in the TKT(+/−) cornea, where TKT is especially abundant in wild-type mice. The small female TKT(+/−) mice mated infrequently and had few progeny (with a male/female ratio of 1.4:1) when pregnant. Thus, TKT in normal mice appears to be carefully balanced at a threshold level for well-being. Our data suggest that TKT deficiency may have clinical significance in humans and raise the possibility that obesity may be treated by partial inhibition of TKT in adipose tissue

Regulation of GSH in ␣A-Expressing Human Lens Epithelial Cell Lines and in ␣A Knockout Mouse Lenses

PURPOSE. To study the mechanism of regulation of GSH in HLE-B3 cells expressing ␣A-crystallin (␣A) and in ␣A knockout mouse lenses. METHODS. GSH levels and maximal rates of GSH synthesis were measured in immortalized, ␣A-transfected HLE-B3 cells containing varying amounts of ␣A. The mRNA and protein for the rate-limiting enzyme for GSH synthesis, ␥-glutamylcysteine synthetase (GCS), were also determined in ␣A-and mock-transfected cells by Northern blot analysis and Western blot analysis of heavy (GCS-HS) and light (GCS-LS) subunits. The effect of absence of ␣A and ␣B on lens GSH concentrations was evaluated in whole lenses of ␣A knockout and ␣B knockout mice as a function of age. GCS-HS mRNA and protein were determined in young, precataractous and cataractous ␣A knockout lenses. RESULTS. GSH levels were significantly higher in HLE-B3 cells expressing ␣A-compared with mock-transfected cells and were correlated positively with ␣A content. Mean rate of GSH synthesis was also higher in ␣A-expressing cells than in mock controls (0.84 vs. 0.61 nmol ⅐ min Ϫ1 per mg protein, respectively). GCS-HS mRNA and GCS-LS mRNA were approximately twofold higher in ␣A-expressing cells, whereas the heavy and light GCS subunit proteins increased by 80% to 100%. In ␣A(Ϫ/Ϫ) mouse lenses, GSH level was not different from that of wild type up to 2 months from birth, after which it dropped to ϳ50% of controls. On the other hand, GCS-HS and GCS-LS proteins showed a significant decrease before cataract formation as early as 15 days after birth. GSH level in cataract-free ␣B(Ϫ/Ϫ) lenses was similar to that of wild type for up to 14 months. CONCLUSIONS. Expression of ␣A caused an increase in cellular GSH, in part, because of an increase in mRNA and protein of both GCS subunits. GSH levels decreased with increasing age in cataractous ␣A(Ϫ/Ϫ) lenses but not in the noncataractous ␣B(Ϫ/Ϫ) lenses. It is suggested that neonatal precataractous lenses (with normal GSH and decreased GCS) may maintain their GSH level by other compensatory mechanisms such as increased GSH transport. (Invest Ophthalmol Vis Sci. 2001;42: 409 -416

Structurally Normal Corneas in Aldehyde Dehydrogenase 3a1-Deficient Mice

We have constructed an ALDH3a1 null mouse to investigate the role of this enzyme that comprises nearly one-half of the total water-soluble protein in the mouse corneal epithelium. ALDH3a1-deficient mice are viable and fertile, have a corneal epithelium with a water-soluble protein content approximately half that of wild-type mice, and contain no ALDH3a1 as determined by zymograms and immunoblots. Despite the loss of protein content and ALDH3a1 activity, the ALDH3a1(−/−) mouse corneas appear indistinguishable from wild-type corneas when examined by histological analysis and electron microscopy and are transparent as determined by light and slit lamp microscopy. There is no evidence for a compensating protein or enzyme. Even though the function of ALDH3a1 in the mouse cornea remains unknown, our data indicate that its enzymatic activity is unnecessary for corneal clarity and maintenance, at least under laboratory conditions