Maria Felice Brizzi

STAT Protein Recruitment and Activation in c-Kit Deletion Mutants*

Stem cell factor (SCF) and its tyrosine kinase receptor, c-Kit, play a crucial role in regulating migration and proliferation of melanoblasts, germ cells, and hemopoietic cell progenitors by activating a number of intracellular signaling molecules. Here we report that SCF stimulation of myeloid cells or fibroblasts ectopically expressing c-Kit induces physical association with and tyrosine phosphorylation of three signal transducers and activators of transcription (STATs) as follows: STAT1α, STAT5A, and STAT5B. Other STAT proteins are not recruited upon SCF stimulation. Recruitment of STATs leads to their dimerization, nuclear translocation, and binding to specific promoter-responsive elements. Whereas STAT1α, possibly in the form of homodimers, binds to thesis-inducible DNA element, STAT5 proteins, either as STAT5A/STAT5B or STAT5/STAT1α heterodimers, bind to the prolactin-inducible element of the β-casein promoter. The tyrosine kinase activity of Kit appears essential for STAT activation since a kinase-defective mutant lacking a kinase insert domain was inactive in STAT signaling. However, another mutant that lacked the carboxyl-terminal region retained STAT1α activation and nuclear translocation but was unable to fully activate STAT5 proteins, although it mediated their transient phosphorylation. These results indicate that different intracellular domains of c-Kit are involved in activation of the various STAT proteins.

IL-3 is a novel target to interfere with tumor vasculature

Angiogenesis inhibiting agents are currently integral component of anticancer therapy. However, tumors, initially responsive to anti-angiogenic drugs or vascular targeting agents, can acquire resistance. The limited clinical efficacy might result from the heterogeneous nature of tumors or alternatively from the unique phenotype of tumor vascular cells, widely diverse from so-called ‘normal’ endothelium. Hence, defining the molecular mechanisms driving this diversity might provide a rational basis to design combinatory therapies that should be more effective in avoiding resistance. Herein, we demonstrated that tumor-derived endothelial cells (TECs) isolated from breast and kidney carcinomas retained an endothelial phenotype, but outspread independently of growth factors. Applying small interfering RNA approach, we demonstrated that interleukin (IL)-3, but not vascular endothelial growth factor, released by TECs, supports their autocrine growth and promotes in vivo vessel formation and tumor angiogenesis. Meanwhile, we found that the expression of the membrane-bound kit ligand (mbKitL) depends on IL-3, and it is crucial for adhesion of endothelial progenitor cells (EPCs) and inflammatory cells to TECs. These events required Akt activation. Finally, the finding that depletion of the mbKitL prevented EPC and inflammatory cell trafficking into vascular microenvironment, indicates that, as in bone marrow, the mbKitL can act as a membrane/adhesion molecule for c-Kit-expressing cells. These data provide evidences that an IL-3 autocrine loop can drive a tumor endothelial switch and that targeting IL-3 might confer a significant therapeutic advantage to hamper tumor angiogenesis.

Inhibition of β1 integrin and IL-3Rβ common subunit interaction hinders tumour angiogenesis.

Integrin/cytokine receptor interaction provides permissive signals leading to neoangiogenesis, and integrins are crucial for differentiation of endothelial progenitor cells (EPCs). It is known that the inflammatory interleukin-3 (IL-3), released in the tumoral microenvironment, contributes to both angiogenesis and vasculogenic processes. Herein, we generated IL-3 receptor beta common (IL-3Rβc) extracellular domain-derived fusion proteins (Fc) to elucidate the molecular mechanisms regulating these processes. Three different Fc were generated, containing the entire extracellular domain of IL-3Rβc (Fc1.4), a fragment corresponding to domains 1–3 (Fc1.3) and a fragment corresponding to domain 4 (Fc4), respectively. The ability of the fusion proteins to interfere with IL-3Rβc/β1 integrin interaction was assessed on endothelial cells (ECs), EPCs and murine-derived ECs. Pull-down experiments showed that Fc1.4 and Fc4 fusion proteins specifically interacted with β1 integrin. Fc4 and Fc1.4 fragments prevented IL-3-mediated EPC expansion, arterial morphogenesis and tumour-derived EC migration, without affecting cell adhesion. Fc4 in vivo inhibited the IL-3-mediated vasculogenic process, as well as inflammatory and tumour vascular growth. In conclusion, these data identify the β1 integrin-interacting domain in the juxta-membrane IL-3Rβc extracellular domain, and provide the rational for targeting this interaction to impair vascular growth.

Polyunsaturated fatty acids and cytokines: Their relationship in acute lung injury

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are inflammatory diseases whose clinical severity depends on the grade of inflammatory response. Inflammatory cytokines are key elements in the pathogenesis of ALI/ARDS, and the occurrence of an imbalance between proand anti-inflammatory cytokines leads to additional non-pulmonary organ dysfunction which contributes to excess mortality rates. Treatment of these patients includes nutrition support with lipids, usually soybean oil-based lipid emulsions, which are rich in omega (n)-6 polyunsaturated fatty acids (PUFAs) and deficient in n-3 PUFAs; however, too much n-6 PUFAs are detrimental due to their pro-inflammatory effects. Conversely, a large amount of experimental studies and some randomized clinical trials showed the benefits of the n-3 PUFA administration in the context of ALI because of their anti-inflammatory properties. Based on these data, several scientific societies recommended in their guidelines, with an A or B grade of recommendation, the use of n-3 PUFAs in ALI/ARDS patients. However, at present, the issue of lipid therapy in ALI/ARDS is still controversial due, at least in part, to inconclusive or contradicting results in several recent clinical trials using n-3 PUFAs. List of Abbreviations AA Arachidonic acid ALI Acute lung injury ARDS Acute respiratory distress syndrome BAL Bronchoalveolar lavage DHA Docosahexaenoic acid EN Enteral nutrition EPA Eicosapentaenoic acid FA Fatty acid FiO2 Fractional inspired oxygen ICU Intensive care unit IL Interleukin LOS Length of stay LPS Lipopolysaccharide LT Leukotriene *Email: paolo.cotogni@unito.it Diet and Nutrition in Critical Care DOI 10.1007/978-1-4614-8503-2_112-1 # Springer Science+Business Media New York 2014