Dominant Noonan syndrome-causing LZTR1 mutations specifically affect the Kelch domain substrate-recognition surface and enhance RAS-MAPK signaling.

Noonan syndrome (NS), the most common RASopathy, is caused by mutations affecting signaling through RAS and the MAPK cascade. Recently, genome scanning has discovered novel genes implicated in NS, whose function in RAS-MAPK signaling remains obscure, suggesting the existence of unrecognized circuits contributing to signal modulation in this pathway. Among these genes, leucine zipper-like transcriptional regulator 1 (LZTR1) encodes a functionally poorly characterized member of the BTB/POZ protein superfamily. Two classes of germline LZTR1 mutations underlie dominant and recessive forms of NS, while constitutional monoallelic, mostly inactivating, mutations in the same gene cause schwannomatosis, a cancer-prone disorder clinically distinct from NS. Here we show that dominant NS-causing LZTR1 mutations do not affect significantly protein stability and subcellular localization. We provide the first evidence that these mutations, but not the missense changes occurring as biallelic mutations in recessive NS, enhance stimulus-dependent RAS-MAPK signaling, which is triggered, at least in part, by an increased RAS protein pool. Moreover, we document that dominant NS-causing mutations do not perturb binding of LZTR1 to CUL3, a scaffold coordinating the assembly of a multimeric complex catalyzing protein ubiquitination but are predicted to affect the surface of the Kelch domain mediating substrate binding to the complex. Collectively, our data suggest a model in which LZTR1 contributes to the ubiquitinationof protein(s) functioning as positive modulator(s) of the RAS-MAPK signaling pathway. In this model, LZTR1 mutations are predicted to variably impair binding of these substrates to the multi-component ligase complex and their efficient ubiquitination and degradation, resulting in MAPK signaling upregulation.

Local and transient inhibition of p21 expression ameliorates age-related delayed wound healing.

Nonhealing chronic wounds in the constantly growing elderly population represent a major public health problem with high socioeconomic burden. Yet, the underlying mechanism of age-related impairment of wound healing remains elusive. Here, we show that the number of dermal cells expressing cyclin-dependent kinase inhibitor p21 was elevated upon skin injury, particularly in aged population, in both man and mouse. The nuclear expression of p21 in activated wound fibroblasts delayed the onset of the proliferation phase of wound healing in a p53-independent manner. Further, the local and transient inhibition of p21 expression by in vivo delivered p21-targeting siRNA ameliorated the delayed wound healing in aged mice. Our results suggest that the increased number of p21+ wound fibroblasts enforces the age-related compromised healing, and targeting p21 creates potential clinical avenues to promote wound healing in aged population.

Increased osteoclastogenesis contributes to bone loss in the Costello syndrome Hras G12V mouse model.

RAS GTPases are ubiquitous GDP/GTP-binding proteins that function as molecular switches in cellular signalling and control numerous signalling pathways and biological processes. Pathogenic mutations in RAS genes severely affect cellular homeostasis, leading to cancer when occurring in somatic cells and developmental disorders when the germline is affected. These disorders are generally termed as RASopathies and among them Costello syndrome (CS) is a distinctive entity that is caused by specific HRAS germline mutations. The majority of these mutations affect residues 12 and 13, the same sites as somatic oncogenic HRAS mutations. The hallmarks of the disease include congenital cardiac anomalies, impaired thriving and growth, neurocognitive impairments, distinctive craniofacial anomalies, and susceptibility to cancer. Adult patients often present signs of premature aging including reduced bone mineral density and osteoporosis. Using a CS mouse model harbouring a Hras G12V germline mutation, we aimed at determining whether this model recapitulates the patients' bone phenotype and which bone cells are driving the phenotype when mutated. Our data revealed that Hras G12V mutation induces bone loss in mice at certain ages. In addition, we identified that bone loss correlated with an increased number of osteoclasts in vivo and Hras G12V mutations increased osteoclastogenesis in vitro. Last, but not least, mutant osteoclast differentiation was reduced by treatment in vitro with MEK and PI3K inhibitors, respectively. These results indicate that Hras is a novel regulator of bone homeostasis and an increased osteoclastogenesis due to Hras G12V mutation contributes to bone loss in the Costello syndrome.

The glucocorticoid receptor associates with RAS complexes to inhibit cell proliferation and tumor growth.

Mutations that activate members of the RAS family of GTPases are associated with various cancers and drive tumor growth. The glucocorticoid receptor (GR), a member of the nuclear receptor family, has been proposed to interact with and inhibit the activation of components of the PI3K-AKT and MAPK pathways downstream of RAS. In the absence of activating ligands, we found that GR was present in cytoplasmic KRAS-containing complexes and inhibited the activation of wild-type and oncogenic KRAS in mouse embryonic fibroblasts and human lung cancer A549 cells. The DNA binding domain of GR was involved in the interaction with KRAS, but GR-dependent inhibition of RAS activation did not depend on the nuclear translocation of GR. The addition of ligand released GR-dependent inhibition of RAS, AKT, the MAPK p38, and the MAPKK MEK. CRISPR-Cas9-mediated deletion of GR in A549 cells enhanced tumor growth in xenografts in mice. Patient samples of non-small cell lung carcinomas showed lower expression of NR3C1, the gene encoding GR, compared to adjacent normal tissues and lower NR3C1 expression correlated with a worse disease outcome. These results suggest that glucocorticoids prevent the ability of GR to limit tumor growth by inhibiting RAS activation, which has potential implications for the use of glucocorticoids in patients with cancer.

Studying Metabolic Abnormalities in the Costello Syndrome HRAS G12V Mouse Model: Isolation of Mouse Embryonic Fibroblasts and Their In Vitro Adipocyte Differentiation.

Costello syndrome (CS), characterized by a developmental delay and a failure to thrive, is also associated with an impaired lipid and energy metabolism. White adipose tissue is a central sensor of whole-body energy homeostasis, and HRAS hyperactivation may affect adipocyte differentiation and mature adipocyte homeostasis. An extremely useful tool for delineating in vitro intrinsic cellular signaling leading to metabolic alterations during adipogenesis is mouse embryonic fibroblasts, known to differentiate into adipocytes in response to adipogenesis-stimulating factors. Here, we describe in detail the isolation and maintenance of CS HRAS G12V mouse embryonic fibroblasts, their differentiation into adipocytes, and an assessment of adipocyte differentiation.

Senescence in RASopathies, a possible novel contributor to a complex pathophenoype.

Senescence is a biological process that induces a permanent cell cycle arrest and a specific gene expression program in response to various stressors. Following studies over the last few decades, the concept of senescence has evolved from an antiproliferative mechanism in cancer (oncogene-induced senescence) to a critical component of physiological processes associated with embryonic development, tissue regeneration, ageing and its associated diseases. In somatic cells, oncogenic mutations in RAS-MAPK pathway genes are associated with oncogene-induced senescence and cancer, while germline mutations in the same pathway are linked to a group of monogenic developmental disorders generally termed RASopathies. Here, we consider that in these disorders, senescence induction may result in opposing outcomes, a tumour protective effect and a possible contributor to a premature ageing phenotype identified in Costello syndrome, which belongs to the RASopathy group. In this review, we will highlight the role of senescence in organismal homeostasis and we will describe the current knowledge about senescence in RASopathies. Additionally, we provide a perspective on examples of experimentally characterised RASopathy mutations that, alone or in combination with various stressors, may also trigger an age-dependent chronic senescence, possibly contributing to the age-dependent worsening of RASopathy pathophenotype and the reduction of lifespan.

Cdk5 Deletion Enhances the Anti-inflammatory Potential of GC-Mediated GR Activation During Inflammation.

The suppression of activated pro-inflammatory macrophages during immune response has a major impact on the outcome of many inflammatory diseases including sepsis and rheumatoid arthritis. The pro- and anti-inflammatory functions of macrophages have been widely studied, whereas their regulation under immunosuppressive treatments such as glucocorticoid (GC) therapy is less well-understood. GC-mediated glucocorticoid receptor (GR) activation is crucial to mediate anti-inflammatory effects. In addition, the anti-cancer drug roscovitine, that is currently being tested in clinical trials, was recently described to regulate inflammatory processes by inhibiting different Cdks such as cyclin-dependent kinase 5 (Cdk5). Cdk5 was identified as a modulator of inflammatory processes in different immune cells and furthermore described to influence GR gene expression in the brain. Whether roscovitine can enhance the immunosuppressive effects of GCs and if the inhibition of Cdk5 affects GR gene regulatory function in innate immune cells, such as macrophages, has not yet been investigated. Here, we report that roscovitine enhances the immunosuppressive Dexamethasone (Dex) effect on the inducible nitric oxide synthase (iNos) expression, which is essential for immune regulation. Cdk5 deletion in macrophages prevented iNos protein and nitric oxide (NO) generation after a combinatory treatment with inflammatory stimuli and Dex. Cdk5 deletion in macrophages attenuated the GR phosphorylation on serine 211 after Dex treatment alone and in combination with inflammatory stimuli, but interestingly increased the GR-dependent anti-inflammatory target gene dual-specificity phosphatase 1 (Dusp1, Mkp1). Mkp1 phosphatase activity decreases the activation of its direct target p38Mapk, reduced iNos expression and NO production upon inflammatory stimuli and Dex treatment in the absence of Cdk5. Taken together, we identified Cdk5 as a potential novel regulator of NO generation in inflammatory macrophages under GC treatment. Our data suggest that GC treatment in combination with specific Cdk5 inhibtior(s) provides a stronger suppression of inflammation and could thus replace high-dose GC therapy which has severe side effects in the treatment of inflammatory diseases.