Pentraxin 3 Facilitates Shrimp-Allergic Responses in IgE-Activated Mast Cells.

Background: Since food avoidance is currently the only way to prevent allergic reactions to shrimp, a better understanding of molecular events in the induction and progression of allergy, including food allergy, is needed for developing strategies to inhibit allergic responses. Pentraxin 3 (PTX3) is rapidly produced directly from inflammatory or damaged tissues and is involved in acute immunoinflammatory responses. However, the role of PTX3 in the development of immediate IgE-mediated shrimp allergy remains unknown. Methods: Wild-type BALB/c mice were immunized intraperitoneally and were challenged with shrimp extract. Serum IgE and PTX3 levels were analyzed. RBL-2H3 cells were stimulated with either dinitrophenyl (DNP) or serum of shrimp-allergic mice, and markers of degranulation, proinflammatory mediators, and phosphorylation of signal proteins were analyzed. We further examined the effect of PTX3 in shrimp extract-induced allergic responses in vitro and in vivo. Results: Mice with shrimp allergy had increased PTX3 levels in the serum and small intestine compared with healthy mice. PTX3 augmented degranulation, the production of proinflammatory mediators, and activation of the Akt and MAPK signaling pathways in mast cells upon DNP stimulation. Furthermore, the expression of transcription factor CCAAT/enhancer-binding protein delta (CEBPD) was elevated in PTX3-mediated mast cell activation. Finally, the PTX3 inhibitor RI37 could attenuate PTX3-induced degranulation, proinflammatory mediator expression, and phosphorylation of the Akt and MAPK signaling. Conclusions: The results suggested that PTX3 can facilitate allergic responses. Our data provide new insight to demonstrate that PTX3 is a cause of allergic inflammation and that RI37 can serve as a therapeutic agent in shrimp allergy.

Disruption of the pentraxin 3/CD44 interaction as an efficient therapy for triple-negative breast cancers.

Due to the heterogeneity and high frequency of genome mutations in cancer cells, targeting vital protumour factors found in stromal cells in the tumour microenvironment may represent an ideal strategy in cancer therapy. However, the regulation and mechanisms of potential targetable therapeutic candidates need to be investigated. An in vivo study demonstrated that loss of pentraxin 3 (PTX3) in stromal cells significantly decreased the metastasis and growth of cancer cells. Clinically, our results indicate that stromal PTX3 expression correlates with adverse prognostic features and is associated with worse survival outcomes in triple-negative breast cancer (TNBC). We also found that transforming growth factor beta 1 (TGF-ß1) induces PTX3 expression by activating the transcription factor CCAAT/enhancer binding protein delta (CEBPD) in stromal fibroblasts. Following PTX3 stimulation, CD44, a PTX3 receptor, activates the downstream ERK1/2, AKT and NF-κB pathways to specifically contribute to the metastasis/invasion and stemness of TNBC MDA-MB-231 cells. Two types of PTX3 inhibitors were developed to disrupt the PTX3/CD44 interaction and they showed a significant effect on attenuating growth and restricting the metastasis/invasion of MDA-MB-231 cells, suggesting that targeting the PTX3/CD44 interaction could be a new strategy for future TNBC therapies.

Suppression of prolactin signaling by pyrrolidine dithiocarbamate is alleviated by N-acetylcysteine in mammary epithelial cells.

Prolactin is the key hormone to stimulate milk synthesis in mammary epithelial cells. It signals through the Jak2-Stat5 pathway to induce the expression of ß-casein, a milk protein which is often used as a marker for mammary differentiation. Here we examined the effect of pyrrolidine dithiocarbamate (PDTC) on prolactin signaling. Our results show that PDTC downregulates prolactin receptor levels, and inhibits prolactin-induced Stat5 tyrosine phosphorylation and ß-casein expression. This is not due to its inhibitory action on NF-κB since application of another NF-κB inhibitor, BAY 11-7082, and overexpression of I-κBα super-repressor do not lead to the same results. Instead, the pro-oxidant activity of PDTC is involved as inclusion of the antioxidant N-acetylcysteine restores prolactin signaling. PDTC triggers great extents of activation of ERK and JNK in mammary epithelial cells. These do not cause suppression of prolactin signaling but confer serine phosphorylation of insulin receptor substrate-1, thereby perturbing insulin signal propagation. As insulin facilitates optimal ß-casein expression, blocking insulin signaling by PDTC might pose additional impediment to ß-casein expression. Our results thus imply that lactation will be compromised when the cellular redox balance is dysregulated, such as during mastitis.

The RhoA-Rok-myosin II pathway is involved in extracellular matrix-mediated regulation of prolactin signaling in mammary epithelial cells.

In mammary epithelial cells (MECs), prolactin-induced signaling and gene expression requires integrin-mediated cell adhesion to basement membrane (BM). In the absence of proper cell-BM interactions, for example, culturing cells on collagen-coated plastic dishes, signal propagation is substantially impaired. Here we demonstrate that the RhoA-Rok-myosin II pathway accounts for the ineffectiveness of prolactin signaling in MECs cultured on collagen I. Under these culture conditions, the RhoA pathway is activated, leading to downregulation of prolactin receptor expression and reduced prolactin signaling. Enforced activation of RhoA in MECs cultured on BM suppresses prolactin receptor levels, and prevents prolactin-induced Stat5 tyrosine phosphorylation and ß-casein expression. Overexpression of dominant negative RhoA in MECs cultured on collagen I, or inhibiting Rok activity, increases prolactin receptor expression, and enhances prolactin signaling. In addition, inhibition of myosin II ATPase activity by blebbistatin also exerts a beneficial effect on prolactin receptor expression and prolactin signaling, suggesting that tension exerted by the collagen substratum, in collaboration with the RhoA-Rok-myosin II pathway, contributes to the failure of prolactin signaling. Furthermore, MECs cultured on laminin-coated plastic have similar morphology and response to prolactin as those cultured on collagen I. They display high levels of RhoA activity and are inefficient in prolactin signaling, stressing the importance of matrix stiffness in signal transduction. Our results reveal that RhoA has a central role in determining the fate decisions of MECs in response to cell-matrix interactions.

Extracellular matrix controls insulin signaling in mammary epithelial cells through the RhoA/Rok pathway.

Cellular responses are determined by a number of signaling cues in the local microenvironment, such as growth factors and extracellular matrix (ECM). In cultures of mammary epithelial cells (MECs), functional differentiation requires at least two types of signal, lactogenic hormones (i.e., prolactin, insulin, and hydrocortisone) and the specialized ECM, basement membrane (BM). Our previous work has shown that ECM affects insulin signaling in mammary cells. Cell adhesion to BM promotes insulin-stimulated tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and association of PI3K with IRS-1, whereas cells cultured on stromal ECM are inefficient in transducing these post-receptor events. Here we examine the mechanisms underlying ECM control of IRS phosphorylation. Compared to cells cultured on BM, cells on plastic exhibit higher level of RhoA activity. The amount and the activity of Rho kinase (Rok) associated with IRS-1 are greater in these cells, leading to serine phosphorylation of IRS-1. Expression of dominant negative RhoA and the application of Rok inhibitor Y27632 in cells cultured on plastic augment tyrosine phosphorylation of IRS-1. Conversely, expression of constitutively active RhoA in cells cultured on BM impedes insulin signaling. These data indicate that RhoA/Rok is involved in substratum-mediated regulation of insulin signaling in MECs, and under the conditions where proper adhesion to BM is missing, such as after wounding and during mammary gland involution, insulin-mediated cellular differentiation and survival would be defective.

TGF-beta inhibits prolactin-induced expression of beta-casein by a Smad3-dependent mechanism.

Transforming growth factor-beta (TGF-beta) is a multifunctional growth factor, affecting cell proliferation, apoptosis, and extracellular matrix homeostasis. It also plays critical roles in mammary gland development, one of which involves inhibition of the expression of milk proteins, such as beta-casein, during pregnancy. Here we further explore the underlying signaling mechanism for it. Our results show that TGF-beta suppresses prolactin-induced expression of beta-casein mRNA and protein in primary mouse mammary epithelial cells, but its effect on protein expression is more evident. We also find out that this inhibition is not due to the effect of TGF-beta on cell apoptosis. Furthermore, inhibition of TGF-beta type I receptor kinase activity by a pharmacological inhibitor SB431542 or overexpression of dominant negative Smad3 substantially restores beta-casein expression. By contrast, inhibition of p38 and Erk that are known to be activated by TGF-beta does not alleviate the inhibitory effect of TGF-beta. These results are consistent with our other observation that Smad but not MAPK pathway is activated by TGF-beta in mammary epithelial cells. Lastly, we show that prolactin-induced tyrosine phosphorylation of Jak2 and Stat5 as well as serine/threonine phosphorylation of p70S6K and S6 ribosomal protein are downregulated by TGF-beta, although the former event requires considerably long exposure to TGF-beta. We speculate that these events might be involved in repressing transcription and translation of beta-casein gene, respectively. Taken together, our results demonstrate that TGF-beta abrogates prolactin-stimulated beta-casein gene expression in mammary epithelial cells through, at least in part, a Smad3-dependent mechanism.