Genetic Analysis of Connective Tissue Growth Factor as an Effector of Transforming Growth Factor β Signaling and Cardiac Remodeling

The matricellular secreted protein connective tissue growth factor (CTGF) is upregulated in response to cardiac injury or with transforming growth factor β (TGF-β) stimulation, where it has been suggested to function as a fibrotic effector. Here we generated transgenic mice with inducible heart-specific CTGF overexpression, mice with heart-specific expression of an activated TGF-β mutant protein, mice with heart-specific deletion of Ctgf, and mice in which Ctgf was also deleted from fibroblasts in the heart. Remarkably, neither gain nor loss of CTGF in the heart affected cardiac pathology and propensity toward early lethality due to TGF-β overactivation in the heart. Also, neither heart-specific Ctgf deletion nor CTGF overexpression altered cardiac remodeling and function with aging or after multiple acute stress stimuli. Cardiac fibrosis was also unchanged by modulation of CTGF levels in the heart with aging, pressure overload, agonist infusion, or TGF-β overexpression. However, CTGF mildly altered the overall cardiac response to TGF-β when pressure overload stimulation was applied. CTGF has been proposed to function as a critical TGF-β effector in underlying tissue remodeling and fibrosis throughout the body, although our results suggest that CTGF is of minimal importance and is an unlikely therapeutic vantage point for the heart.     

Genetic Variations in Key MicroRNA Processing Genes and Risk of Head and Neck Cancer: A Case-Control Study in Chinese Population

MicroRNAs (miRNAs) have been reported to play a key role in oncogenesis. Genetic variations in miRNA processing genes and miRNA binding sites may affect the biogenesis of miRNA and the miRNA-mRNA interactions, hence promoting tumorigenesis. In the present study, we hypothesized that potentially functional polymorphisms in miRNA processing genes may contribute to head and neck cancer (HNC) susceptibility. To test this hypothesis, we genotyped three SNPs at miRNA binding sites of miRNA processing genes (rs1057035 in 3′UTR of DICER, rs3803012 in 3′UTR of RAN and rs10773771 in 3′UTR of HIWI) with a case-control study including 397 HNC cases and 900 controls matched by age and sex in Chinese. Although none of three SNPs was significantly associated with overall risk of HNC, rs1057035 in 3′UTR of DICER was associated with a significantly decreased risk of oral cancer (TC/CC vs. TT: adjusted OR  = 0.65, 95% CI  = 0.46–0.92). Furthermore, luciferase activity assay showed that rs1057035 variant C allele led to significantly lower expression levels as compared to the T allele, which may be due to the relatively high inhibition of hsa-miR-574-3p on DICER mRNA. These findings indicated that rs1057035 located at 3′UTR of DICER may contribute to the risk of oral cancer by affecting the binding of miRNAs to DICER. Large-scale and well-designed studies are warranted to validate our findings.     

Triplex forming oligonucleotide targeted to 3'UTR downregulates the expression of the bcl-2 proto-oncogene in HeLa cells

The bcl-2 proto-oncogene is overexpressed in a variety of human cancers and plays an important role in programmed cell death. Recent reports implied that the 3′-untranslated region (3′UTR) functions effectively in the regulation of gene expression. Here, we attempt to assay the ability of triplex forming oligonucleotides (TFOs) to inhibit expression of a target gene in vivo and to examine the potential of the 3′UTR of the bcl-2 proto-oncogene in the regulation of bcl-2 gene expression. To do this, we have developed a novel cellular system that involves transfection of a Doxycyclin inducible expression plasmid containing the bcl-2 ORF and the 3′UTR together with a TFO targeted to the 3′UTR of the bcl-2 proto-oncogene. Phosphorothioate-modified TFO targeted to the 3′UTR of the bcl-2 gene significantly downregulated the expression of the bcl-2 gene in HeLa cells as demonstrated by western blotting. Our results indicate that blocking the functions of the 3′UTR using the TFO can downregulate the expression of the targeted gene, and suggest that triplex strategy is a promising approach for oligonucleotide-based gene therapy. In addition, triplex-based sequence targeting may provide a useful tool for studying the regulation of gene expression.     

Allele Specific Expression of the Transthyretin Gene in Swedish Patients with Hereditary Transthyretin Amyloidosis (ATTR V30M) Is Similar between the Two Alleles

Background

Hereditary transthyretin (TTR) amyloidosis (ATTR) is an autosomal dominant disease characterized by extracellular deposits of amyloid fibrils composed of misfolded TTR. The differences in penetrance and age at onset are vast, both between and within populations, with a generally late onset for Swedish carriers. In a recent study the entire TTR gene including the 3′ UTR in Swedish, French and Japanese ATTR patients was sequenced. The study disclosed a SNP in the V30M TTR 3′ UTR of the Swedish ATTR population that was not present in either the French or the Japanese populations (rs62093482-C>T). This SNP could create a new binding site for miRNA, which would increase degradation of the mutated TTR’s mRNA thus decrease variant TTR formation and thereby delay the onset of the disease. The aim of the present study was to disclose differences in allele specific TTR expression among Swedish V30M patients, and to see if selected miRNA had any effect upon the expression.

Methodology/Principal Findings

Allele-specific expression was measured on nine liver biopsies from Swedish ATTR patients using SNaPshot Multiplex assay. Luciferase activity was measured on cell lines transfected with constructs containing the TTR 3′ UTR. Allele-specific expression measured on liver biopsies from Swedish ATTR patients showed no difference in expression between the two alleles. Neither was there any difference in expression between cell lines co-transfected with two constructs with or without the TTR 3′ UTR SNP regardless of added miRNA.

Conclusions/Significance

The SNP found in the 3′ UTR of the TTR gene has no effect on degrading the variant allele’s expression and thus has no impact on the diminished penetrance of the trait in the Swedish population. However, the 3′ UTR SNP is unique for patients descending from the Swedish founder, and this SNP could be utilized to identify ATTR patients of Swedish descent.     

CTGF Mediates Smad-Dependent Transforming Growth Factor β Signaling To Regulate Mesenchymal Cell Proliferation during Palate Development

Transforming growth factor β (TGF-β) signaling plays crucial functions in the regulation of craniofacial development, including palatogenesis. Here, we have identified connective tissue growth factor (Ctgf) as a downstream target of the TGF-β signaling pathway in palatogenesis. The pattern of Ctgf expression in wild-type embryos suggests that it may be involved in key processes during palate development. We found that Ctgf expression is downregulated in both Wnt1-Cre; Tgfbr2^fl/fl and Osr2-Cre; Smad4^fl/fl palates. In Tgfbr2 mutant embryos, downregulation of Ctgf expression is associated with p38 mitogen-activated protein kinase (MAPK) overactivation, whereas loss of function of Smad4 itself leads to downregulation of Ctgf expression. We also found that CTGF regulates its own expression via TGF-β signaling. Osr2-Cre; Smad4^fl/fl mice exhibit a defect in cell proliferation similar to that of Tgfbr2 mutant mice, as well as cleft palate. We detected no alteration in bone morphogenetic protein (BMP) downstream targets in Smad4 mutant palates, suggesting that the reduction in cell proliferation is due to defective transduction of TGF-β signaling via decreased Ctgf expression. Significantly, an exogenous source of CTGF was able to rescue the cell proliferation defect in both Tgfbr2 and Smad4 mutant palates. Collectively, our data suggest that CTGF regulates proliferation as a mediator of the canonical pathway of TGF-β signaling during palatogenesis.     

Regulation of the Stability of Heat-Stable Antigen mRNA by Interplay between Two Novel cis Elements in the 3′ Untranslated Region

The heat-stable antigen (HSA) is a costimulatory molecule for T-cell activation. Its expression is strictly regulated during lymphocyte development and differentiation. Recent studies using HSA-transgenic mice have demonstrated that this regulated expression is critical for normal development of T and B lymphocytes. However, the mechanisms that control the expression of HSA are largely unknown. HSA mRNA is comprised of a 0.23-kb open reading frame and a 1.5-kb 3′ untranslated region (3′UTR). The function of the long 3′UTR has not been addressed. Here we investigate the role of the 3′UTR of HSA mRNA. We show that a 160-bp element, located in the region of nucleotides 1465 to 1625 in the 3′UTR of HSA mRNA, promotes RNA degradation and that this effect is neutralized by a 43-bp fragment approximately 1 kb upstream of the negative cis element. Both positive and negative cis elements in the HSA mRNA are distinct from other sequences that are known to modulate mRNA stability. These results provide direct evidence that the interplay between two novel cis elements in the 3′UTR of HSA mRNA determines cell surface HSA expression by modulating its RNA stability.     

Sequences within Both the 5′ UTR and Gag Are Required for Optimal In Vivo Packaging and Propagation of Mouse Mammary Tumor Virus (MMTV) Genomic RNA

Background

This study mapped regions of genomic RNA (gRNA) important for packaging and propagation of mouse mammary tumor virus (MMTV). MMTV is a type B betaretrovirus which preassembles intracellularly, a phenomenon distinct from retroviruses that assemble the progeny virion at cell surface just before budding such as the type C human and feline immunodeficiency viruses (HIV and FIV). Studies of FIV and Mason-Pfizer monkey virus (MPMV), a type D betaretrovirus with similar intracellular virion assembly processes as MMTV, have shown that the 5′ untranslated region (5′ UTR) and 5′ end of gag constitute important packaging determinants for gRNA.

Methodology

Three series of MMTV transfer vectors containing incremental amounts of gag or 5′ UTR sequences, or incremental amounts of 5′ UTR in the presence of 400 nucleotides (nt) of gag were constructed to delineate the extent of 5′ sequences that may be involved in MMTV gRNA packaging. Real time PCR measured the packaging efficiency of these vector RNAs into MMTV particles generated by co-transfection of MMTV Gag/Pol, vesicular stomatitis virus envelope glycoprotein (VSV-G Env), and individual transfer vectors into human 293T cells. Transfer vector RNA propagation was monitored by measuring transduction of target HeLaT4 cells following infection with viral particles containing a hygromycin resistance gene expression cassette on the packaged RNA.

Principal Findings

MMTV requires the entire 5′ UTR and a minimum of ∼120 nucleotide (nt) at the 5′ end of gag for not only efficient gRNA packaging but also propagation of MMTV-based transfer vector RNAs. Vector RNAs without the entire 5′ UTR were defective for both efficient packaging and propagation into target cells.

Conclusions/Significance

These results reveal that the 5′ end of MMTV genome is critical for both gRNA packaging and propagation, unlike the recently delineated FIV and MPMV packaging determinants that have been shown to be of bipartite nature.