Changes in the vascular smooth muscle cell (VSMC) contractile phenotype occur in pathological states such as restenosis and atherosclerosis. Multiple cytokines, signaling through receptor tyrosine kinases (RTK) and PI3K/Akt and MAPK/ERK pathways, regulate these phenotypic transitions. The Spry proteins are feedback modulators of RTK signaling, but their specific roles in VSMC have not been established. Here, we report for the first time that Spry1, but not Spry4, is required for maintaining the differentiated state of human VSMC in vitro. While Spry1 is a known MAPK/ERK inhibitor in many cell types, we found that Spry1 has little effect on MAPK/ERK signaling but increases and maintains Akt activation in VSMC. Sustained Akt signaling is required for VSMC marker expression in vitro, while ERK signaling negatively modulates Akt activation and VSMC marker gene expression. Spry4, which antagonizes both MAPK/ERK and Akt signaling, suppresses VSMC differentiation marker gene expression. We show using siRNA knockdown and ChIP assays that FoxO3a, a downstream target of PI3K/Akt signaling, represses myocardin promoter activity, and that Spry1 increases, while Spry4 decreases myocardin mRNA levels. Together, these data indicate that Spry1 and Spry4 have opposing roles in VSMC phenotypic modulation, and Spry1 maintains the VSMC differentiation phenotype in vitro in part through an Akt/FoxO/myocardin pathway.

Interleukin-17 receptor D (IL17RD or IL-17RD) also known as Sef (similar expression to fibroblast growth factor), is a single pass transmembrane protein that is reported to regulate several signaling pathways . IL17RD was initially described as a feedback inhibitor of fibroblast growth factor (FGF) signaling during zebrafish and frog development. It was subsequently determined to regulate other receptor tyrosine kinase signaling cascades as well as several proinflammatory signaling pathways including Interleukin-17A (IL17A), Toll-like receptors (TLR) and Interleukin-1α (IL1α) in several vertebrate species including humans. This review will provide an overview of IL17RD regulation of signaling pathways and functions with emphasis on regulation of development and pathobiological conditions. We will also discuss gaps in our knowledge about IL17RD function to provide insight into opportunities for future investigation.

Background: Growth factors and their receptors are mediators of organogenesis and must be tightly regulated in a temporal and spatial manner for proper tissue morphogenesis. Intracellular regulators of growth factor signaling pathways provide an additional level of control. Members of the Sprouty family negatively regulate receptor tyrosine kinase pathways in several developmental contexts. To gain insight into the role of Spry1 in neural crest development, we analyzed the developmental effects of conditional expression of Spry1 in neural crest-derived tissues. Results: Here we report that conditional expression of Spry1 in neural crest cells causes defects in craniofacial and cardiac development in mice. Spry1;Wnt1-Cre embryos die perinatally and exhibit facial clefting, cleft palate, cardiac and cranial nerve defects. These defects appear to be the result of decreased proliferation and increased apoptosis of neural crest and neural crest-derived cell populations. In addition, the domains of expression of several key transcription factors important to normal craniofacial and cardiac development including AP2, Msx2, Dlx5, and Dlx6 were reduced in Spry1;Wnt1-Cre transgenic embryos. Conclusion: Collectively, these data suggest that Spry1 is an important regulator of craniofacial and cardiac morphogenesis and perturbations in Spry1 levels may contribute to congenital disorders involving tissues of neural crest origin.

Sprouty (Spry) proteins have been implicated in cancer progression, but their role in triple-negative breast cancer (TNBC), a subtype of lethal and aggressive breast cancer, is unknown. Here, we reported that Spry1 is significantly expressed in TNBC specimen and MDA-MB-231 cells. To understand Spry1 regulation of signaling events controlling breast cancer phenotype, we used lentiviral delivery of human Spry1 shRNAs to suppress Spry1 expression in MDA-MB-231, an established TNBC cell line. Spry1 knockdown MDA-MB-231 cells displayed an epithelial phenotype with increased membrane E-cadherin expression. Knockdown of Spry1 impaired MDA-MB-231 cell migration, Matrigel invasion, and anchorage-dependent and -independent growth. Tumor xenografts originating from Spry1 knockdown MDA-MB-231 cells grew slower, had increased E-cadherin expression, and yielded fewer lung metastases compared to control. Furthermore, suppressing Spry1 in MDA-MB-231 cells impaired the induction of Snail and Slug expression by EGF, and this effect was associated with increased EGFR degradation and decreased EGFR/Grb2/Shp2/Gab1 signaling complex formation. The same phenotype was also observed in the TNBC cell line MDA-MB-157. Together, our results show that unlike in some tumors, where Spry may mediate tumor suppression, Spry1 plays a selective role in at least a subset of TNBC to promote the malignant phenotype via enhancing EGF-mediated mesenchymal phenotype.

Cancer stem cells contribute to tumor initiation, heterogeneity, and recurrence, and are critical targets in cancer therapy. Sprouty4 (Spry4) is a potent inhibitor of signal transduction pathways elicited by receptor tyrosine kinases, and has roles in regulating cell proliferation, migration and differentiation. Spry4 has been implicated as a tumor suppressor and in modulating embryonic stem cells. The purpose of this research was to test the novel idea that Spry4 regulates cancer stem cell properties in breast cancer. Loss-of function of Spry4 in human MDA-MB-231 cell was used to test our hypothesis. Spry4 knockdown or control cell lines were generated using lentiviral delivery of human Spry4 or non-targeting control shRNAs, and then selected with 2 μg/ml puromycin. Cell growth and migratory abilities were determined using growth curve and cell cycle flow cytometry analyses and scratch assays, respectively. Xenograft tumor model was used to determine the tumorigenic activity and metastasis in vivo. Cancer stem cell related markers were evaluated using immunoblotting assays and fluorescence-activated cell sorting. Cancer stem cell phenotype was evaluated using in vitro mammosphere formation and drug sensitivity tests, and in vivo limiting dilution tumor formation assay. Two out of three tested human Spry4 shRNAs significantly suppressed the expression of endogenous Spry4 in MDA-MB-231 cells. Suppressing Spry4 expression increased MDA-MB-231 cell proliferation and migration. Suppressing Spry4 increased β3-integrin expression, and CD133+CD44+ subpopulation. Suppressing Spry4 increased mammosphere formation, while decreasing the sensitivity of MDA-MB-231 cells to Paclitaxel treatment. Finally, suppressing Spry4 increased the potency of MDA-MB-231 cell tumor initiation, a feature attributed to cancer stem cells. Our findings provide novel evidence that endogenous Spry4 may have tumor suppressive activity in breast cancer by suppressing cancer stem cell properties in addition to negative effects on tumor cell proliferation and migration.

Perivascular adipose tissue (PVAT) regulates vascular function by secreting vasoactive substances. In mice, Notch signaling is activated in the PVAT during diet-induced obesity, and leads to the loss of the thermogenic phenotype and adipocyte whitening due to increased lipid accumulation. We used the Adiponectin-Cre (Adipoq-Cre) strain to activate a ligand-independent Notch1 intracellular domain transgene (N1ICD) to drive constitutive Notch signaling in the adipose tissues (N1ICD;Adipoq-Cre). We previously found that constitutive activation of Notch1 signaling in the PVAT phenocopied the effects of diet-induced obesity. To understand the downstream pathways activated by Notch signaling, we performed a proteomic analysis of the PVAT from control versus N1ICD;Adipoq-Cre mice. This comparison identified prominent changes in the protein signatures related to metabolism, adipocyte homeostasis, mitochondrial function, and ferroptosis. PVAT-derived stromal vascular fraction cells were derived from our mouse strains to study the cellular and molecular phenotypes during adipogenic induction. We found that cells with activated Notch signaling displayed decreased mitochondrial respiration despite similar levels of adipogenesis and mitochondrial number. We observed variable regulation of the proteins related to mitochondrial dynamics and ferroptosis, including PHB3, PINK1, pDRP1, and the phospholipid hydroperoxidase GPX4. Mitochondria regulate some forms of ferroptosis, which is a regulated process of cell death driven by lipid peroxidation. Accordingly, we found that Notch activation promoted lipid peroxidation and ferroptosis in PVAT-derived adipocytes. Because the PVAT phenotype is a regulator of vascular reactivity, we tested the effect of Notch activation in PVAT on vasoreactivity using wire myography. The aortae from the N1ICD;Adipoq-Cre mice had increased vasocontraction and decreased vasorelaxation in a PVAT-dependent and age-dependent manner. Our data provide support for the novel concept that increased Notch signaling in the adipose tissue leads to PVAT whitening, impaired mitochondrial function, increased ferroptosis, and loss of a protective vasodilatory signal. Our study advances our understanding of how Notch signaling in adipocytes affects mitochondrial dynamics, which impacts vascular physiology.