Axin pathway activity regulates in vivo pY654-ß-catenin accumulation and pulmonary fibrosis.

Epithelial to mesenchymal transition (EMT) and pulmonary fibrogenesis require epithelial integrin α3ß1-mediated cross-talk between TGFß1 and Wnt signaling pathways. One hallmark of this cross-talk is pY654-ß-catenin accumulation, but whether pY654-ß-catenin is a biomarker of fibrogenesis or functionally important is unknown. To clarify further the role of ß-catenin in fibrosis, we explored pY654-ß-catenin generation and function. α3ß1 was required for TGFß1-mediated activation of Src family kinases, and Src inhibition blocked both pY654 and EMT in primary alveolar epithelial cells (AECs). TGFß1 stimulated ß-catenin/Lef1-dependent promoter activity comparably in immortalized AECs stably expressing WT ß-catenin as well as Y654E or Y654F ß-catenin point mutants. But EMT was abrogated in the Tyr to Phe mutant. pY654-ß-catenin was sensitive to the axin ß-catenin turnover pathway as inhibition of tankyrase 1 led to high AEC axin levels, loss of pY654-ß-catenin, and inhibition of EMT ex vivo. Mice given a tankyrase inhibitor (50 mg/kg orally) daily for 7 days beginning 10 days after intratracheal bleomycin had improved survival over controls. Treated mice developed raised axin levels in the lung that abrogated pY654-ß-catenin and attenuated lung Snail1, Twist1, α-smooth muscle actin, and type I collagen accumulation. Total ß-catenin levels were unaltered. These findings identify Src kinase(s) as a mediator of TGFß1-induced pY654-ß-catenin, provide evidence that pY654-ß-catenin levels are a critical determinant of EMT and fibrogenesis, and suggest regulation of axin levels as a novel therapeutic approach to fibrotic disorders.

Oxiris Membrane in Sepsis and Multiple Organ Failure.

Sepsis and multiple organ failure (MOF) are characterized by multiple hemodynamic changes and imbalanced immune response of the patient. Oxiris is a highly adsorptive membrane with the ability to remove cytokines and endotoxins, as well as to perform renal replacement therapy. Here we describe the evolution from previous AN69 to the 3-in-1 Oxiris membrane, and review its characteristics and performance. In clinical practice, Oxiris showed consistent effects in mean arterial pressure recovery, a decrease in vasopressor needs, and reduction of the Sequential Organ Failure Assessment score. These results have been reproduced by several independent studies addressing both sepsis and, to a lesser extent, COVID-19 patients. In addition, more recent studies in sepsis showed improvements in MOF duration and the length of stay in the ICU, as well as some promising results regarding mortality. Finally, we review ongoing clinical trials and discuss its potential significance to clinical practice improvement and to further reinforce knowledge on the use of blood purification in sepsis and acute kidney injury.

Regulation of Claspin by the p38 stress-activated protein kinase protects cells from DNA damage.

Stress-activated protein kinases (SAPKs) enhance survival in response to environmental changes. In yeast, the Hog1 SAPK and Mrc1, a protein required for DNA replication, define a safeguard mechanism that allows eukaryotic cells to prevent genomic instability upon stress during S-phase. Here we show that, in mammals, the p38 SAPK and Claspin-the functional homolog of Mrc1-protect cells from DNA damage upon osmostress during S-phase. We demonstrate that p38 phosphorylates Claspin and either the mutation of the p38-phosphorylation sites in Claspin or p38 inhibition suppresses the protective role of Claspin on DNA damage. In addition, wild-type Claspin but not the p38-unphosphorylatable mutant has a protective effect on cell survival in response to cisplatin treatment. These findings reveal a role of Claspin in response to chemotherapeutic drugs. Thus, this pathway protects S-phase integrity from different insults and it is conserved from yeast to mammals.

Functional Network Analysis Reveals the Relevance of SKIIP in the Regulation of Alternative Splicing by p38 SAPK.

Alternative splicing is a prevalent mechanism of gene regulation that is modulated in response to a wide range of extracellular stimuli. Stress-activated protein kinases (SAPKs) play a key role in controlling several steps of mRNA biogenesis. Here, we show that osmostress has an impact on the regulation of alternative splicing (AS), which is partly mediated through the action of p38 SAPK. Splicing network analysis revealed a functional connection between p38 and the spliceosome component SKIIP, whose depletion abolished a significant fraction of p38-mediated AS changes. Importantly, p38 interacted with and directly phosphorylated SKIIP, thereby altering its activity. SKIIP phosphorylation regulated AS of GADD45α, the upstream activator of the p38 pathway, uncovering a negative feedback loop involving AS regulation. Our data reveal mechanisms and targets of SAPK function in stress adaptation through the regulation of AS.

Conserved regulatory motifs in osteogenic gene promoters integrate cooperative effects of canonical Wnt and BMP pathways.

Osteoblast differentiation depends on the coordinated network of evolutionary conserved transcription factors during bone formation and homeostasis. Evidence indicates that bone morphogenetic protein (BMP) and Wnt proteins regulate several steps of skeletal development. Here, we provide a molecular description of the cooperative effects of BMP and Wnt canonical pathway on the expression of the early osteogenic genes Dlx5, Msx2, and Runx2 in C2C12 cells, primary cultures of bone marrow-mesenchymal stem cells, and organotypic calvarial cultures. Coordinated regulation of these genes leads to the cooperative activation of their downstream osteogenic target gene osterix. Induction of these genes is mediated through enhancer regions with an evolutionary conserved structure encompassing both Smad and TCF/LEF1 DNA-binding sites. Formation of a cooperative complex is mediated through DNA binding of Smads and TCF4/ß-catenin to their cognate sequences, as well as protein-protein interactions between them. The formation of these cooperative transcriptional complexes results in a more efficient recruitment of coactivators such as p300. We propose that evolutionary conserved regulatory regions in specific osteogenic master genes are key integrative modules during osteogenesis.

BMP-2 induces Osterix expression through up-regulation of Dlx5 and its phosphorylation by p38.

Osterix, a zinc-finger transcription factor, is specifically expressed in osteoblasts and osteocytes of all developing bones. Because no bone formation occurs in Osterix null mice, Osterix is thought to be an essential regulator of osteoblast differentiation. We report that bone morphogenetic protein-2 (BMP-2) induces an increase in Osterix expression, which is mediated through a homeodomain sequence located in the proximal region of the Osterix promoter. Our results demonstrate that induction of Dlx5 by BMP-2 mediates Osterix transcriptional activation. First, BMP-2 induction of Dlx5 precedes the induction of Osterix. Second, Dlx5 binds to the BMP-responsive homeodomain sequences both in vitro and in vivo. Third, Dlx5 overexpression and knock-down assays demonstrate its role in activating Osterix expression in response to BMP-2. Furthermore, we show that Dlx5 is a novel substrate for p38 MAPK in vitro and in vivo and that Ser-34 and Ser-217 are the sites phosphorylated by p38. Phosphorylation at Ser-34/217 increases the transactivation potential of Dlx5. Thus, we propose that BMP activates expression of Osterix through the induction of Dlx5 and its further transcriptional activation by p38-mediated phosphorylation.