Rodrigo Fernandez-Valdivia

The RANKL signaling axis is sufficient to elicit ductal side-branching and alveologenesis in the mammary gland of the virgin mouse.

Receptor of Activated NF-kappaB Ligand (RANKL) is implicated as one of a number of effector molecules that mediate progesterone and prolactin signaling in the murine mammary epithelium. Using a mouse transgenic approach, we demonstrate that installation of the RANKL signaling axis into the mammary epithelium results in precocious ductal side-branching and alveologenesis in the virgin animal. These morphological changes occur due to RANKL-induced mammary epithelial proliferation, which is accompanied by increases in expression of activated NF-kB and cyclin D1. With age, prolonged RANKL exposure elicits limited mammary epithelial hyperplasia. While these transgenics exhibit RANKL-induced salivary gland adenocarcinomas, palpable mammary tumors are not observed due to RANKL-suppression of its own signaling receptor (RANK) in the mammary epithelium. Together, these studies reveal not only that the RANKL signaling axis can program many of the normal epithelial changes attributed to progesterone and prolactin action in the normal mammary gland during early pregnancy, but underscore the necessity for tight control of this signaling molecule to avoid unwarranted developmental changes that could lead to mammary hyperplasia in later life.

Steroid receptor coactivator 2 is critical for progesterone-dependent uterine function and mammary morphogenesis in the mouse

ABSTRACT Although the essential involvement of the progesterone receptor (PR) in female reproductive tissues is firmly established, the coregulators preferentially enlisted by PR to mediate its physiological effects have yet to be fully delineated. To further dissect the roles of members of the steroid receptor coactivator (SRC)/p160 family in PR-mediated reproductive processes in vivo, state-of-the-art cre-loxP engineering strategies were employed to generate a mouse model (PRCre/+SRC-2flox/flox) in which SRC-2 function was abrogated only in cell lineages that express the PR. Fertility tests revealed that while ovarian activity was normal, PRCre/+SRC-2flox/flox mouse uterine function was severely compromised. Absence of SRC-2 in PR-positive uterine cells was shown to contribute to an early block in embryo implantation, a phenotype not shared by SRC-1 or -3 knockout mice. In addition, histological and molecular analyses revealed an inability of the PRCre/+SRC-2flox/flox mouse uterus to undergo the necessary cellular and molecular changes that precede complete P-induced decidual progression. Moreover, removal of SRC-1 in the PRCre/+SRC-2flox/flox mouse uterus resulted in the absence of a decidual response, confirming that uterine SRC-2 and -1 cooperate in P-initiated transcriptional programs which lead to full decidualization. In the case of the mammary gland, whole-mount and histological analysis disclosed the absence of significant ductal side branching and alveologenesis in the hormone-treated PRCre/+SRC-2flox/flox mammary gland, reinforcing an important role for SRC-2 in cellular proliferative changes that require PR. We conclude that SRC-2 is appropriated by PR in a subset of transcriptional cascades obligate for normal uterine and mammary morphogenesis and function.

Regulation of mammalian Notch signaling and embryonic development by the protein O -glucosyltransferase Rumi

Protein O-glucosylation is a conserved post-translational modification that occurs on epidermal growth factor-like (EGF) repeats harboring the C1-X-S-X-P-C2 consensus sequence. The Drosophila protein O-glucosyltransferase (Poglut) Rumi regulates Notch signaling, but the contribution of protein O-glucosylation to mammalian Notch signaling and embryonic development is not known. Here, we show that mouse Rumi encodes a Poglut, and that Rumi−/− mouse embryos die before embryonic day 9.5 with posterior axis truncation and severe defects in neural tube development, somitogenesis, cardiogenesis and vascular remodeling. Rumi knockdown in mouse cell lines results in cellular and molecular phenotypes of loss of Notch signaling without affecting Notch ligand binding. Biochemical, cell culture and cross-species transgenic experiments indicate that a decrease in Rumi levels results in reduced O-glucosylation of Notch EGF repeats, and that the enzymatic activity of Rumi is key to its regulatory role in the Notch pathway. Genetic interaction studies show that removing one copy of Rumi in a Jag1+/− (jagged 1) background results in severe bile duct morphogenesis defects. Altogether, our data indicate that addition of O-glucose to EGF repeats is essential for mouse embryonic development and Notch signaling, and that Jag1-induced signaling is sensitive to the gene dosage of the protein O-glucosyltransferase Rumi. Given that Rumi−/− embryos show more severe phenotypes compared to those displayed by other global regulators of canonical Notch signaling, Rumi is likely to have additional important targets during mammalian development.

Role of Glycans and Glycosyltransferases in the Regulation of Notch Signaling

The evolutionarily conserved Notch signaling pathway plays broad and important roles during embryonic development and in adult tissue homeostasis. Unlike most other pathways used during animal development, Notch signaling does not rely on second messengers and intracellular signaling cascades. Instead, pathway activation results in the cleavage of the Notch intracellular domain and its translocation into the nucleus, where it functions as a transcriptional co-activator of the Notch target genes. To ensure tight spatial and temporal regulation of a pathway with such an unusually direct signaling transduction, animal cells have devised a variety of specialized modulatory mechanisms. One such mechanism takes advantage of decorating the Notch extracellular domain with rare types of O-linked glycans. In this review, we will discuss the genetic and biochemical data supporting the notion that carbohydrate modification is essential for Notch signaling and attempt to provide a brief historical overview of how we have learned what we know about the glycobiology of Notch. We will also summarize what is known about the contribution of specific nucleotide-sugar transporters to Notch biology and the roles-enzymatic and non-enzymatic-played by specific glycosyltransferases in the regulation of this pathway. Mutations in the Notch pathway components cause a variety of human diseases, and manipulation of Notch signaling is emerging as a powerful tool in regenerative medicine. Therefore, studying how sugar modification modulates Notch signaling provides a framework for better understanding the role of glycosylation in animal development and might offer new tools to manipulate Notch signaling for therapeutic purposes.

Rumi functions as both a protein O-glucosyltransferase and a protein O-xylosyltransferase.

Mutations in rumi result in a temperature-sensitive loss of Notch signaling in Drosophila. Drosophila Rumi is a soluble, endoplasmic reticulum-retained protein with a CAP10 domain that functions as a protein O-glucosyltransferase. In human and mouse genomes, three potential Rumi homologues exist: one with a high degree of identity to Drosophila Rumi (52%), and two others with lower degrees of identity but including a CAP10 domain (KDELC1 and KDELC2). Here we show that both mouse and human Rumi, but not KDELC1 or KDELC2, catalyze transfer of glucose from UDP-glucose to an EGF repeat from human factor VII. Similarly, human Rumi, but not KDELC1 or KDELC2, rescues the Notch phenotypes in Drosophila rumi clones. During characterization of the Rumi enzymes, we noted that, in addition to protein O-glucosyltransferase activity, both mammalian and Drosophila Rumi also showed significant protein O-xylosyltransferase activity. Rumi transfers Xyl or glucose to serine 52 in the O-glucose consensus sequence () of factor VII EGF repeat. Surprisingly, the second serine (S53) facilitates transfer of Xyl, but not glucose, to the EGF repeat by Rumi. EGF16 of mouse Notch2, which has a diserine motif in the consensus sequence (), is also modified with either O-Xyl or O-glucose glycans in cells. Mutation of the second serine (S590A) causes a loss of O-Xyl but not O-glucose at this site. Altogether, our data establish dual substrate specificity for the glycosyltransferase Rumi and provide evidence that amino acid sequences of the recipient EGF repeat significantly influence which donor substrate (UDP-glucose or UDP-Xyl) is used.

Multiple O-glucosylation sites on Notch function as a buffer against temperature-dependent loss of signaling

Mutations in Drosophila rumi result in a temperature-sensitive loss of Notch signaling. Rumi is a protein O-glucosyltransferase that adds glucose to EGF repeats with a C-X-S-X-P-C consensus sequence. Eighteen of the 36 EGF repeats in the Drosophila Notch receptor contain the consensus O-glucosylation motif. However, the contribution of individual O-glucose residues on Notch to the regulation of Notch signaling is not known. To address this issue, we carried out a mutational analysis of these glucosylation sites and determined their effects on Notch activity in vivo. Our results indicate that even though no single O-glucose mutation causes a significant decrease in Notch activity, all of the glucose residues on Notch contribute in additive and/or redundant fashions to maintain robust signaling, especially at higher temperatures. O-glucose motifs in and around the ligand-binding EGF repeats play a more important role than those in other EGF repeats of Notch. However, a single O-glucose mutation in EGF12 can be compensated by other O-glucose residues in neighboring EGF repeats. Moreover, timecourse cell aggregation experiments using a rumi null cell line indicate that a complete lack of Rumi does not affect Notch-Delta binding at high temperature. In addition, rumi fully suppresses the gain-of-function phenotype of a ligand-independent mutant form of Notch. Our data suggest that, at physiological levels of Notch, the combined effects of multiple O-glucose residues on this receptor allow productive S2 cleavage at high temperatures and thereby serve as a buffer against temperature-dependent loss of Notch signaling.

A mouse model to dissect progesterone signaling in the female reproductive tract and mammary gland.

Considering the regulatory complexities of progesterone receptor (PR) action throughout the female reproductive axis and mammary gland, we generated a mouse model that enables conditional ablation of PR function in a spatiotemporal specific manner. Exon 2 of the murine PR gene was floxed to generate a conditional PR allele (PRflox) in mice. Crossing the PRflox/flox mouse with the ZP3‐cre transgenic demonstrated that the PRflox allele recombines to a PR null allele (PRd). Mice homozygous for the recombined null PR allele (PRd/d) exhibit uterine, ovarian, and mammary gland defects that phenocopy those of our previously described PR knockout (PRKO) model. Therefore, this conditional mouse model for PR ablation represents an invaluable resource with which to further define in a developmental and/or reproductive stage‐specific manner the individual and integrative roles of distinct PR populations resident in multiple progesterone‐responsive target sites. genesis 48:106–113, 2010.

Jagged1 heterozygosity in mice results in a congenital cholangiopathy which is reversed by concomitant deletion of one copy of Poglut1 (Rumi)

Haploinsufficiency for the Notch ligand JAG1 in humans results in an autosomal‐dominant, multisystem disorder known as Alagille syndrome, which is characterized by a congenital cholangiopathy of variable severity. Here, we show that on a C57BL/6 background, jagged1 heterozygous mice (Jag1+/−) exhibit impaired intrahepatic bile duct (IHBD) development, decreased SOX9 expression, and thinning of the periportal vascular smooth muscle cell (VSMC) layer, which are apparent at embryonic day 18 and the first postnatal week. In contrast, mice double heterozygous for Jag1 and the glycosyltransferase, Poglut1 (Rumi), start showing a significant improvement in IHBD development and VSMC differentiation during the first week. At P30, Jag1+/− mice show widespread ductular reactions and ductopenia in liver and a mild, but statistically, significant bilirubinemia. In contrast, P30 Jag1/Rumi double‐heterozygous mice show well‐developed portal triads around most portal veins, with no elevation of serum bilirubin. Conditional deletion of Rumi in VSMCs results in progressive arborization of the IHBD tree, whereas deletion of Rumi in hepatoblasts frequently results in an increase in the number of hepatic arteries without affecting bile duct formation. Nevertheless, removing one copy of Rumi from either VSMCs or hepatoblasts is sufficient to partially suppress the Jag1+/− bile duct defects. Finally, all Rumi target sites of the human JAG1 are efficiently glucosylated, and loss of Rumi in VSMCs results in increased levels of full‐length JAG1 and a shorter fragment of JAG1 without affecting Jag1 messenger RNA levels. Conclusions: On a C57BL/6 background, Jag1 haploinsufficiency results in bile duct paucity in mice. Removing one copy of Rumi suppresses the Jag1+/− bile duct phenotype, indicating that Rumi opposes JAG1 function in the liver. (Hepatology 2016;63:550–565)

From the ranks of mammary progesterone mediators, RANKL takes the spotlight

Whether during the diestrus phase of the estrous cycle or with pregnancy onset, the mitogenic effects of progesterone are well-established in the murine mammary epithelium. Importantly, progesterone-induced mitogenicity is critical for mammary tumor promotion, providing one explanation for the increase in breast cancer-risk observed with prolonged progestin-based hormone therapy. At the cellular level, progesterone projects its mitogenic influence through an evolutionary conserved paracrine mechanism of action. In this regard, recent studies provide compelling support for receptor activator of NF-kB ligand (RANKL) as a key paracrine mediator of the progesterone mitogenic signal. Induction of RANKL is sufficient to elicit mammary ductal side-branching and alveologenesis, the very morphogenetic responses elicited by progesterone during pregnancy and at diestrus. Significantly, the proliferative and pro-survival signals triggered by RANKL are also required for progestin-promotion of mammary tumorigenesis, underscoring a dual role for RANKL in progesterone-dependent mammary morphogenesis and tumorigenesis. Recently, RANKL has been shown to be critical for progesterone-induced expansion of the mammary stem cell population (and its lineal descendents), thereby advancing our conceptual understanding not only of RANKLs involvement in normal mammary morphogenesis but also in breast cancer risk associated with sustained hormone exposure. Finally, these studies together suggest that chemotherapeutic intervention of RANKL signaling represents a feasible approach for the effective prevention and/or treatment of hormone-responsive breast cancers.

Steroid receptor coactivator 2 is essential for progesterone-dependent uterine function and mammary morphogenesis: insights from the mouse--implications for the human.

While the indispensability of the progesterone receptor (PR) in female reproduction and mammary morphogenesis is acknowledged, the coregulators preferentially recruited by PR to mediate its in vivo effects have yet to be fully delineated. To further parse the roles of steroid receptor coactivator (SRC)/p160 family members in P-dependent physiological processes, genetic approaches were employed to generate a mouse model (PR(Cre/+)SRC-2(flox/flox)) in which SRC-2 function was ablated specifically in cell-types that express the PR. Fertility evaluation revealed that while ovulation occurred normally in the PR(Cre/+)SRC-2(flox/flox) mouse, uterine function was markedly affected. Absence of SRC-2 in PR positive uterine cells contributed to an early block in embryo implantation, a phenotype not shared by knockouts for SRC-1 or -3. Although the PR(Cre/+)SRC-2(flox/flox) uterus could mount a partial decidual response, removal of SRC-1 in the PR(Cre/+)SRC-2(flox/flox) uterus resulted in a complete block in decidualization, confirming that uterine SRC-2 and -1 are both required for P-initiated transcriptional programs which lead to full decidualization. In the case of the mammary gland, whole-mount and histological analyses revealed the absence of significant branching morphogenesis in the hormone-treated PR(Cre/+)SRC-2(flox/flox) mammary gland, reinforcing an important role for mammary SRC-2 in cellular proliferative events that require PR. Based on the above and the observation that SRC-2 is expressed in many of the uterine and mammary cell-lineages in the human as observed in the mouse, we suggest that further investigations are warranted to gain additional insights into SRC-2s involvement in normal (and possibly abnormal) uterine and mammary cellular responses to progestins.