Modeling autosomal recessive cutis laxa type 1C in mice reveals distinct functions for Ltbp-4 isoforms

Recent studies have revealed an important role for LTBP-4 in elastogenesis. Its mutational inactivation in humans causes autosomal recessive cutis laxa type 1C (ARCL1C), which is a severe disorder caused by defects of the elastic fiber network. Although the human gene involved in ARCL1C has been discovered based on similar elastic fiber abnormalities exhibited by mice lacking the short Ltbp-4 isoform (Ltbp4S(-/-)), the murine phenotype does not replicate ARCL1C. We therefore inactivated both Ltbp-4 isoforms in the mouse germline to model ARCL1C. Comparative analysis of Ltbp4S(-/-) and Ltbp4-null (Ltbp4(-/-)) mice identified Ltbp-4L as an important factor for elastogenesis and postnatal survival, and showed that it has distinct tissue expression patterns and specific molecular functions. We identified fibulin-4 as a previously unknown interaction partner of both Ltbp-4 isoforms and demonstrated that at least Ltbp-4L expression is essential for incorporation of fibulin-4 into the extracellular matrix (ECM). Overall, our results contribute to the current understanding of elastogenesis and provide an animal model of ARCL1C.Peer reviewe

Modeling autosomal recessive cutis laxa type 1C in mice reveals distinct functions for Ltbp-4 isoforms

Recent studies have revealed an important role for LTBP-4 in elastogenesis. Its mutational inactivation in humans causes autosomal recessive cutis laxa type 1C (ARCL1C), which is a severe disorder caused by defects of the elastic fiber network. Although the human gene involved in ARCL1C has been discovered based on similar elastic fiber abnormalities exhibited by mice lacking the short Ltbp-4 isoform (Ltbp4S(-/-)), the murine phenotype does not replicate ARCL1C. We therefore inactivated both Ltbp-4 isoforms in the mouse germline to model ARCL1C. Comparative analysis of Ltbp4S(-/-) and Ltbp4-null (Ltbp4(-/-)) mice identified Ltbp-4L as an important factor for elastogenesis and postnatal survival, and showed that it has distinct tissue expression patterns and specific molecular functions. We identified fibulin-4 as a previously unknown interaction partner of both Ltbp-4 isoforms and demonstrated that at least Ltbp-4L expression is essential for incorporation of fibulin-4 into the extracellular matrix (ECM). Overall, our results contribute to the current understanding of elastogenesis and provide an animal model of ARCL1C.Peer reviewe

Latent Transforming Growth Factor β-Binding Protein 4 Is Downregulated in Esophageal Cancer via Promoter Methylation

<div><p>Latent transforming growth factor β-binding protein 4 (LTBP4) is an extracellular matrix molecule that is a member of important connective tissue networks and is needed for the correct folding and the secretion of TGF-β1. LTBP4 is downregulated in carcinomas of various tissues. Here we show that LTBP4 is also downregulated in adenocarcinomas and squamous cell carcinomas of the esophagus <i>in vitro</i> and <i>in vivo</i>. Re-expression of LTBP4 in esophageal cancer cell lines reduced cell migration ability, whereas cell viability and cell proliferation remained unchanged. Hypermethylation of the promoter regions of the two main human LTBP4 transcriptional forms, LTBP4L and LTBP4S, was found to be involved in LTBP4 silencing. Detailed investigations of the methylation patterns of the promoter regions of LTBP4L and LTBP4S identified GATA1, SP1, E2F4 and SMAD3 as potential transcription factors involved in LTBP4 expression. In <i>in vitro</i> transcription factor activity studies we discovered E2F4 as novel powerful regulator for LTBP4S expression.</p></div

Demethylation treatment with 5-aza-2’-deoxycytidine induces LTBP4 expression, whereas TGF-β1 expression is not changed.

<p>A) qPCR analysis showed induction of LTBP4 expression in OE33 and KYSE180 cells after demethylation treatment with 5-aza-2’-deoxycytidine. B) qPCR analysis showed no significant changes in TGF-β1 expression in OE33 and KYSE180 cells after demethylation treatment with 5-aza-2’-deoxycytidine. Untreated cells served as control. Experiments were repeated at least 3 times and the mean value was calculated. Data are presented ± standard deviation and *p<0.05 versus control.</p

Re-expression of LTBP4 leads to less migration ability of esophageal carcinoma cells.

<p>A) Migration distance of OE33 and in KYSE180 cells after transient transfection with either pcDNA6 as a control or pcDNA6-LTBP4 within 24 h. Experiments were repeated at least 3 times. The symbols represent the results of each individual experiment and the graphs document the respective mean values. Significances are given: ^#p = n.s. and *p<0.05. B) Representative immunofluorescence staining of the migration assay performed with KYSE180 cells re-expressing LTBP4. LTBP4 (green) and c-myc (red) positive cells (yellow) defined the borders of the cell free gap. Only non-transfected cells migrated into the cell free gap. Nuclei were counterstained with DAPI (blue). The arrow indicates the migration direction. Bar graph: 50 µm.</p

Identification of highly methylated binding sites for transcription factors in the LTBP4L and LTBP4S promoter.

<p><i>In silico</i> analysis of LTBP4 promoter sites identified a highly methylated binding site for GATA1 in the LTBP4L promoter of OE33 and KYSE180 cells. Highly methylated binding sites for SP1 and E2F4 are found in the LTBP4S promoter of both cell lines. A binding site for SMAD3 was identified nearby. The methylation status was analyzed by clonal bisulfite sequencing and at least ten clones were sequenced for each cell line. The percentage of methylation is visualized as pie chart. Exons are illustrated by black boxes and LTBP4S and LTBP4L promoters by red lines. The translation start sites are indicated (ATG). The dashed lines represent alternative splicing of LTBP4.</p

Methylation status of the two predicted LTBP4 promoter regions in esophageal carcinoma cell lines.

<p>The methylation status in the putative LTBP4 promoter regions was analyzed by clonal bisulfite sequencing in OE33 and KYSE180 cells. Eight CpG islands were identified and at least ten clones were sequenced for each cell line. The percentage of methylation in each CpG island is visualized as pie chart. Exons are illustrated by black boxes and LTBP4 promoters by red lines. The translation start sites are indicated (ATG). The dashed lines represent alternative splicing of LTBP4.</p

Effect of Gata1, SP1, SMAD3 and E2F4 on transcriptional activity of LTBP4L and LTBP4S promoter.

<p>HEK293 cells were transiently transfected with pGL4.10-LTBP4L or pGL4.10-LTBP4S and a <i>Renilla</i> luciferase expression vector for normalization. Cells were also co-transfected with different concentrations of A) a Gata1 expression vector, B) a SP1 expression vector, C) a SMAD3 expression vector or D) an E2F4 expression vector. After 24 h cells were lysed and luciferase activities were determined. Experiments were repeated at least 3 times and the mean value was calculated. Data are presented ± standard deviation.</p

Function of Ltbp-4L and fibulin-4 in survival and elastogenesis in mice

LTBP-4L and LTBP-4S are two isoforms of the extracellular matrix protein latent-transforming growth factor beta-binding protein 4 (LTBP-4). The mutational inactivation of both isoforms causes autosomal recessive cutis laxa type1C(ARCL1C) in humans and an ARCL1C-like phenotype in Ltbp4(-/-) mice, both characterized by high postnatal mortality and severely affected elastogenesis. However, genetic data in mice suggest isoform-specific functions for Ltbp-4 because Ltbp4S(-/-) mice, solely expressing Ltbp-4L, survive to adulthood. This clearly suggests a requirement of Ltbp-4L for postnatal survival. A major difference between Ltbp4S(-/-) and Ltbp4(-/-) mice is the matrix incorporation of fibulin-4 (a key factor for elastogenesis; encoded by the Efemp2 gene), which is normal in Ltbp4S(-/-) mice, whereas it is defective in Ltbp4(-/-) mice, suggesting that the presence of Ltbp-4L might be required for this process. To investigate the existence of a functional interaction between Ltbp-4L and fibulin-4, we studied the consequences of fibulin-4 deficiency in mice only expressing Ltbp-4L. Resulting Ltbp4S(-/-); Fibulin-4R/R mice showed a dramatically reduced lifespan compared to Ltbp4S(-/-) or Fibulin-4R/R mice, which survive to adulthood. This dramatic reduction in survival of Ltbp4S(-/-); Fibulin-4R/R mice correlates with severely impaired elastogenesis resulting in defective alveolar septation and distal airspace enlargement in lung, and increased aortic wall thickness with severely fragmented elastic lamellae. Additionally, Ltbp4S(-/-); Fibulin-4R/R mice suffer from aortic aneurysm formation combined with aortic tortuosity, in contrast to Ltbp4S(-/-) or Fibulin-4R/R mice. Together, in accordance with our previous biochemical findings of a physical interaction between Ltbp-4L and fibulin-4, these novel in vivo data clearly establish a functional link between Ltbp-4L and fibulin-4 as a crucial molecular requirement for survival and elastogenesis in mice

Opposing roles of Akt and STAT3 in the protection of the maternal heart from peripartum stress

Background Peripartum cardiomyopathy ( PPCM) is a pregnancy-associated cardiomyopathy in previously healthy women. Mice with a cardiomyocyte-restricted deletion of signal transducer and activator of transcription-3 ( STAT3, CKO) develop PPCM. PI3K-Akt signalling is thought to promote cardiac hypertrophy and protection during pregnancy. We evaluated the role of activated Akt signalling in the maternal heart postpartum. Methods and results CKOmice were bred to mice harbouring an Akt transgene, specifically expressed in cardiomyocytes ( CAkt(tg)) generating CKO; CAkt(tg), CAkttg, CKO, and wild-type sibling mice. CAkt(tg) and CKO; CAkttg female mice developed PPCM with systolic dysfunction. Both genotypes displayed cardiac hypertrophy and lower capillary density, showed increased phosphorylation of p66 Src homology 2 domain containing protein and FoxO3A, and reduced expression of manganese superoxide dismutase as well as increased cathepsin D activity and increased miR-146a levels [ indicative for generation of the anti-angiogenic 16 kDa prolactin ( PRL)]. Cardiac inflammation and fibrosis was accelerated in CKO; CAkt(tg) and associated with high postpartum mortality. The PRL blocker, bromocriptine ( BR), prevented heart failure and the decrease in capillary density in CKO; CAkt(tg) and CAkt(tg) mice. BR attenuated high mortality, up-regulation of CCL2, and cardiac inflammation as well as fibrosis in CKO; CAkt(tg). PRL infusion induced cardiac inflammation in CKO; CAkt(tg) independent of pregnancy. In neonatal rat cardiomyocytes, PRL and interferon g ( IFN gamma) induced the expression of CCL2 via activation of Akt. Conclusion Postpartum Akt activation is detrimental for the peripartum heart as it lowers anti-oxidative defence and in combination with low STAT3 conditions, accelerate cardiac inflammation and fibrosis. PRL and its cleaved 16 kDa form are central for Akt-induced PPCM as indicated by the protection from the disease by PRL blockade