RLIM enhances BMP signalling mediated fetal lung development in mice

RLIM, also called RNF12, is an X-linked E3 ubiquitin ligase. It is a pleotropic protein acting as a negative regulator of LIM-homeodomain transcription factors and of SMAD7, an antagonist of the BMP and TGFβ pathways. Mutations in RLIM are associated with variable congenital malformations in human. The molecular mechanism causing these malformations is still poorly understood. In this study, we used a conventional knockout (KO) mouse model to phenotypically characterize male mutants and to investigate the effect of Rlim mutation on BMP pathway signalling. Only 50% of KO male neonates survive and are significantly smaller. We show that Rlim deletion alters lung branching and maturation resulting in severe respiratory distress and neonatal death. Except for a single case of cardiac interventricular septal defect, no other malformations that mimic the human condition were observed. Western blot analysis shows that RLIM deficiency is associated with an increase of its target SMAD7 levels and a loss of SMAD1/5/9 activation. Interestingly, SMAD5 levels were diminished, suggesting another regulatory loop between RLIM and the BMP pathway. In conclusion, RLIM is important for lung development in mice and exerts this function in part through intracellular propagation of BMP signalling. Author summary RLIM is an X-linked gene encoding an enzyme that regulates a number of proteins including SMAD7, an antagonist of BMP pathway. RLIM mutations are associated with intellectual disability and several congenital malformations in boys. In mice, RLIM is thought to be nonessential for embryos development. Using a mouse model in which Rlim is deleted, we show that 50% of males carrying the deletion display respiratory distress and die short after birth. No congenital malformations mimicking the human phenotype have been observed. Histological analysis of lung samples show that Rlim is critical for late lung development and maturation. The analysis of lung samples by western blot, a technique that quantifies proteins, is consistent with a dysregulation of BMP pathway. These finding confirm the importance of RLIM for murine embryos development and provide a link between human RLIM pathology and BMP pathway.


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RLIM, also called RNF12, is an X-linked gene that encodes a widely expressed RING-H2 zinc 32 finger protein acting as an E3 ubiquitin ligase [1]. RLIM was first discovered as a LIM  [7]. The male lethality was not the main focus of these studies and was not investigated. 54 However, Shin

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Rlim deletion causes a growth delay and neonatal lethality 91 To determine the distribution of the Rlim (Rnf12) deletion, 19 crosses of Rnf12 -/y KO males and 92 Rnf12 +/+ WT females (in the −/+ or +/− nomenclature, the maternally inherited allele is shown 93 first), and 25 crosses of heterozygous Rnf12 +/females and Rnf12 +/y WT males were performed.  To better define the lethality stage, we dissected embryos at E9.5 and at E16.5. All but two 103 Rnf12 -/+ females died before E16.5. The female phenotype will not be discussed in this paper.

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As expected for males, numbers of WT Rnf12 +/y and Rnf12 -/y KO male E9.5 embryos were not We therefore proceeded to dissections at E18.5. Following cesarian section, a total of 56 feti 113 were monitored for 1 hour and were weighed. All the pups were viable as indicated by a beating 114 heart at the extraction moment. Surprisingly, all but one Rnf12 -/y pup (16/17) showed respiratory 115 failure signs: cyanosis, gasps with costal retraction, and deceased within 30 minutes (n=16).

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Given the paleness of KO lungs, we investigated the pulmonary vascular development using  prior to lung extraction and their genotype was confirmed by immunoblotting using an anti-153 RLIM antibody (Fig 7A). We analyzed canonical BMP effector signaling Phosphorylated 154 SMAD1/5/9, the BMP pathway antagonist SMAD7, and the BMP target gene ID3.

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Phosphorylated SMAD1/5/9 was drastically reduced in KO lung samples (Fig 7A). suggesting that ID3 is also regulated by non-SMAD mediated signaling. As expected from the 158 absence of RLIM, SMAD7 was increased in KO males, and this increase was found to be 159 statistically different (Figs 7A, 8).
10 160 Next, to further understand the molecular mechanism underlying this defective BMP signaling 161 and since SMAD1 and its target effector WIF1 (WNT Inhibitory Factor I) are involved in lung 162 maturation [20], we measured the unphosphorylated SMAD1 and SMAD5 and WIF1 levels. 163 Given the variability of these proteins' level, 6 more WT littermates were analyzed by western 164 blot analysis. Surprisingly, whilst SMAD5 was significantly reduced in KOs, SMAD1 and 165 WIF1 were equally expressed in both groups (Figs 7 B, 8).  However, RLIM biological function in mammalian organogenesis has never been explored.

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Using lung lysates, we addressed RLIM involvement in BMP signaling and confirmed the 205 positive correlation between endogenous RLIM level and BMP activity as indicated by an 206 increase of SMAD7 and phosphorylated SMAD1/5/9 depletion in RLIM deficient lungs.

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The lung phenotype resulting from Rlim knockout resembles the phenotypes that have 208 previously been reported in studies exploring BMP pathway involvement in lung development. activate BMP pathway through SMAD7 degradation, but also through interaction with 221 SMURF1 (Fig 9). SMAD5 reduction could result from a decreased RLIM-SMURF1 interaction 222 and therefore an enhanced SMURF1 activity, either induced or not by accumulated SMAD7.    R package was used for statistical calculations. Binomial test (Fig 1) and Mann-Whitney U 320 test (Figs 3, 5 and 6) were used to determine statistical significance. A p-value below 0.05 321 was considered statistically significant.