IntroductionMyeloid cell leukemia sequence 1 (Mcl-1) 1 has been identified as an intracellular antiapoptotic factor in a variety of hematopoietic cells, both in vitro and in vivo. [2][3][4][5][6] Human mast cells express 7,8 and Mcl-1 can promote the survival of some populations of human neoplastic mast cells in vitro. 7 Basophils, granulocytes with many characteristics and functions that partially overlap with those of tissue mast cells, [9][10][11][12] can also express Mcl-1. 13 However, it is not clear to what extent Mcl-1 is important in the development and/or survival of mast cells or basophils in vivo.Opferman et al showed that the genetic manipulation of Mcl-1 can be used to delete individual hematopoietic cell populations in mice. 4 We therefore used this approach to examine the effects of reducing expression of Mcl-1 in the mast cell lineage in vivo. To attempt to delete Mcl-1 selectively in mast cells, we used the promoter for the peptidase carboxypeptidase A3 (CPA3; originally named mast cell carboxypeptidase A 14 ). CPA3 is highly expressed in mast cells, 15 but is also expressed in basophils 16 and can be expressed in some populations of T-cell progenitors and thymic T cells 17,18 and in certain hematopoietic progenitor cells. 19 We generated C57BL/6 mice in which a segment of the Cpa3 promoter drives expression of Cre recombinase, and then mated these Cpa3-Cre transgenic mice to mice bearing a floxed allele of Mcl-1. 4 We found that C57BL/6-Cpa3-Cre; Mcl-1 fl/fl mice are severely deficient in mast cells and have a marked deficiency in basophils, and also exhibit striking impairment in mast cell-or basophil-and IgE-dependent biologic responses. Methods MiceAll animal experiments were carried out following protocols approved by the Stanford University Administrative Panel on Laboratory Animal Care. B6-Tg(Cpa3-cre)3Glli (Cpa3-Cre-transgenic mice) were generated by microinjecting the Cpa3-Cre transgene into embryonic stem cells in the B6 background (Stanford University). Gt(ROSA)26Sor tm4(ACTB-tdTomato,-EGFP)Luo /J(mT/mG) mice, obtained from The Jackson Laboratory, were crossed to Cpa3-Cre mice for Cre expression analysis. Mcl-1 ϩ/fl (B6;129-Mcl1 tm3sjk J) animals were obtained from The Jackson Laboratory. Mcl-1 ϩ/fl mice were bred to progeny from 2 Cpa3-Cre founder lines (founder lines #4 and #5) to obtain Cpa3-Cre; Mcl-1 ϩ/ϩ , Cpa3-Cre; Mcl-1 ϩ/fl , and Cpa3-Cre; Mcl-1 fl/fl animals, but only the Cpa3-Cre; Mcl-1 fl/fl mice derived from founder line #4 exhibited a substantial mast cell deficiency. Therefore, the mice used were derived from crosses between founder line #4 (subsequently referred to as Cpa3-Cre mice) and Mcl-1 fl animals, and these mice had been intercrossed a minimum of 6 generations into the C57BL/6 background. Heterozygous Cpa3-Cre mice were determined to have 5 copies of the Cpa3-Cre transgene by real-time PCR. To emphasize that Cpa3-Cre; Mcl-1 fl/fl mice have deficiencies in mast cells and basophils that are independent of mutations affecting Kit, we call them informally in our labo...

Simultaneous ablation of the two known activators of plasminogen (Plg), urokinase-type (uPA) and the tissuetype (tPA), results in a substantial delay in skin wound healing. However, wound closure and epidermal re-epithelialization are significantly less impaired in uPA;tPA double-deficient mice than in Plg-deficient mice. Skin wounds in uPA;tPA-deficient mice treated with the broad-spectrum matrix metalloproteinase (MMP) inhibitor galardin (N-[(2R)-2-(hydroxamido-carbonylmethyl)-4-methylpentanoyl]-L-tryptophan methylamide) eventually heal, whereas skin wounds in galardin-treated Plg-deficient mice do not heal. Furthermore, plasmin is biochemically detectable in wound extracts from uPA;tPA double-deficient mice. In vivo administration of a plasma kallikrein (pKal)-selective form of the serine protease inhibitor ecotin exacerbates the healing impairment of uPA;tPA double-deficient wounds to a degree indistinguishable from that observed in Plgdeficient mice, and completely blocks the activity of pKal, but not uPA and tPA in wound extracts. These findings demonstrate that an additional plasminogen activator provides sufficient plasmin activity to sustain the healing process albeit at decreased speed in the absence of uPA, tPA and galardin-sensitive MMPs and suggest that pKal plays a role in plasmin generation.

The stromal microenvironment regulates mammary gland branching morphogenesis. We have observed that mast cells are present in the mammary gland throughout its postnatal development and in particular, are found around the terminal end buds and ductal epithelium of the pubertal gland. Mast cells contribute to allergy, inflammatory diseases, and cancer development, but have not been implicated in normal development. Genetic and pharmacological disruption of mast cell function in the mammary gland revealed that mast cells are involved in rapid proliferation and normal duct branching during puberty, and this effect is independent of macrophage recruitment, which also regulates mammary gland development. For mast cells to exert their effects on normal morphogenesis required activation of their serine proteases and degranulation. Our observations reveal a novel role for mast cells during normal pubertal development in the mammary gland.

Obesity has become a prevalent health hazard in industrialized countries and is closely associated with a number of pathological disorders such as cancer, cardiovascular disease, hypertension, and non-insulin-dependent diabetes. 1 Even with such serious health implications there is still a great deal to learn before we can fully understand the mechanisms controlling adipocyte differentiation during normal development as well as in pathological conditions. Adipose tissues are aggregates of specially differentiated mesenchymal cells capable of storing large amounts of triglycerides in periods of energy excess and subsequently releasing those triglycerides during energy deprivation. Kawaguchi and colleagues 2 in this issue of The American Journal of Pathology introduce a mouse model that, together with several other recent studies, 3,4 may lead to greater understanding of the events in the extracellular microenvironment that mediate mesenchymal fate decisions and adipocyte differentiation.The adipose lineage arises from a multipotent stem cell of mesenchymal origin from which muscle, bone, and cartilage cells are also derived, although the exact combination of regulatory genes that determine adipocyte fate in development is yet to be determined. 5 In most species, white adipose tissue formation begins before birth and expands rapidly after birth as a result of increased fat cell size as well as an increase in fat cell number. It is not clear whether the increased adipose tissue mass of obesity is because of increases in adipocyte size or number, or a combination thereof. 6 The availability of established preadipocyte cell lines, such as 3T3-L1 and 3T3-F442A, has made it possible to investigate the adipocyte differentiation program under controlled conditions. Through in vitro studies the transcriptional mechanisms that control the transition from the undifferentiated, fibroblast-like preadipocyte into mature, round fat cells are now well understood, particularly with respect to the identification of key transcription factors such as those of the C/EBP and PPAR families. 5,7 Although most studies have been devoted to the hormonal and transcriptional regulation of adipocyte differentiation, new attention is being given to the role of extracellular matrix (ECM) organization in adipogenesis.During adipogenesis ECM remodeling defines the onset of the differentiation process; this is characterized by the conversion from the fibronectin-rich stromal matrix of the preadipocyte to the basement membrane of an adipocyte ( Figure 1). 3,8,9 The expression of ECM components is highly regulated during the process of adipocyte differentiation: types I and III collagen, fibronectin, and ␤1-integrins are down-regulated, whereas type IV collagen and entactin are up-regulated. 5 This ECM remodeling is a key event in the adipogenesis program. Growth of preadipocytes on a fibronectin matrix inhibits adipocyte differentiation, and this effect is overcome by the addition of cytochalasin D, which disrupts actin filaments and promotes roundi...

The plasminogen cascade of serine proteases directs both development and tumorigenesis in the mammary gland. Plasminogen can be activated to plasmin by urokinase-type plasminogen activator (uPA), tissue-type plasminogen activator (tPA), and plasma kallikrein (PKal). The dominant plasminogen activator for mammary involution is PKal, a serine protease that participates in the contact activation system of blood coagulation. We observed that the prekallikrein gene (Klkb1) is expressed highly in the mammary gland during stromal remodeling periods including puberty and postlactational involution. We used a variant of ecotin (ecotin-PKal), a macromolecular inhibitor of serine proteases engineered to be highly specific for active PKal, to demonstrate that inhibition of PKal with ecotinPKal delays alveolar apoptosis, adipocyte replenishment, and stromal remodeling in the involuting mammary gland, producing a phenotype resembling that resulting from plasminogen deficiency. Using biotinylated ecotin-PKal, we localized active PKal to connective tissue-type mast cells in the mammary gland. Taken together, these results implicate PKal as an effector of the plasminogen cascade during mammary development.The plasminogen cascade of serine proteases regulates both development and tumorigenesis in the mammary gland (1, 2). The ultimate effector in this cascade, plasminogen as its active form, plasmin, is mediated by an intricate cascade of plasminogen activators and protease inhibitors. Plasminogen-deficient mice exhibit significant defects in lactational competence and post-lactational mammary gland involution (2), the process by which the differentiated, lactating gland remodels after the cessation of lactation to a state approaching that of the non-pregnant animal. The effect of plasminogen loss is exacerbated after a round of pregnancy and lactation: plasminogen-null mammary glands have poorly developed secretory alveoli during lactation, and upon involution, never fully involute. Instead, the secretory alveoli fail to regress normally. Moreover, the stroma becomes fibrotic and is cleared incompletely of partially degraded epithelial basement membrane. Because plasminogen-deficient mice largely are unable to support a second round of pregnancy and lactation (2), this suggests that the involution defect is not overcome by activities of other proteases eventually. These studies establish plasminogen as a crucial protease in normal mammary gland biology.Plasminogen is synthesized in the liver and circulates as a zymogen through blood plasma to all vascularized tissues of the body. As this expression and circulation are constant,

Nerve growth factor (NGF) binds to its cognate receptor TrkA and induces neuronal differentiation by activating distinct downstream signal transduction events. RabGEF1 (also known as Rabex-5) is a guanine nucleotide exchange factor for Rab5, which regulates early endosome fusion and vesicular trafficking in endocytic pathways. Here, we used the antisense (AS) expression approach to induce an NGF-dependent sustained knockdown of RabGEF1 protein expression in stable PC12 transfectants. We show that RabGEF1 is a negative regulator of NGF-induced neurite outgrowth and modulates other cellular and signaling processes that are activated by the interaction of NGF with TrkA receptors, such as cell cycle progression, cessation of proliferation, and activation of NGF-mediated downstream signaling responses. Moreover, RabGEF1 can bind to Rac1, and the activation of Rac1 upon NGF treatment is significantly enhanced in AS transfectants, suggesting that RabGEF1 is a negative regulator of NGF-induced Rac1 activation in PC12 cells. Furthermore, we show that RabGEF1 can also interact with NMDA receptors by binding to the NR2B subunit and its associated binding partner SynGAP, and negatively regulates activation of nitric oxide synthase activity induced by NMDA receptor stimulation in NGF-differentiated PC12 cells. Our data suggest that RabGEF1 is a negative regulator of TrkA-dependent neuronal differentiation and of NMDA receptor-mediated signaling activation in NGF-differentiated PC12 cells.

Zena Werb was a legendary scientist and remarkable human being who made defining contributions to the study of nearly every mammalian organ and stage of development and myriad human diseases. She had an encyclopedic knowledge of past literature and an open, creative orientation toward the future. She was a direct and unfiltered critic, a dear friend, a supportive mentor, and a pioneering advocate for women. She was born during World War II and raised by refugee parents in the midst of bigotry, racism, and xenophobia, yet she lived her life as an optimist. We remember her with a smile on her face-a knowing oneinformed by hard experience. We start, as she would want, with the science.

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