Marina Schorpp-Kistner

AP-1 subunits: quarrel and harmony among siblings

The AP-1 transcription factor is mainly composed of Jun, Fos and ATF protein dimers. It mediates gene regulation in response to a plethora of physiological and pathological stimuli, including cytokines, growth factors, stress signals, bacterial and viral infections, as well as oncogenic stimuli. Studies in genetically modified mice and cells have highlighted a crucial role for AP-1 in a variety of cellular events involved in normal development or neoplastic transformation causing cancer. However, emerging evidence indicates that the contribution of AP-1 to determination of cell fates critically depends on the relative abundance of AP-1 subunits, the composition of AP-1 dimers, the quality of stimulus, the cell type and the cellular environment. Therefore, AP-1-mediated regulation of processes such as proliferation, differentiation, apoptosis and transformation should be considered within the context of a complex dynamic network of signalling pathways and other nuclear factors that respond simultaneously.

Altered endochondral bone development in matrix metalloproteinase 13-deficient mice

The assembly and degradation of extracellular matrix (ECM) molecules are crucial processes during bone development. In this study, we show that ECM remodeling is a critical rate-limiting step in endochondral bone formation. Matrix metalloproteinase (MMP) 13 (collagenase 3) is poised to play a crucial role in bone formation and remodeling because of its expression both in terminal hypertrophic chondrocytes in the growth plate and in osteoblasts. Moreover, a mutation in the human MMP13 gene causes the Missouri variant of spondyloepimetaphyseal dysplasia. Inactivation of Mmp13 in mice through homologous recombination led to abnormal skeletal growth plate development. Chondrocytes differentiated normally but their exit from the growth plate was delayed. The severity of the Mmp13- null growth plate phenotype increased until about 5 weeks and completely resolved by 12 weeks of age. Mmp13-null mice had increased trabecular bone, which persisted for months. Conditional inactivation of Mmp13 in chondrocytes and osteoblasts showed that increases in trabecular bone occur independently of the improper cartilage ECM degradation caused by Mmp13 deficiency in late hypertrophic chondrocytes. Our studies identified the two major components of the cartilage ECM, collagen type II and aggrecan, as in vivo substrates for MMP13. We found that degradation of cartilage collagen and aggrecan is a coordinated process in which MMP13 works synergistically with MMP9. Mice lacking both MMP13 and MMP9 had severely impaired endochondral bone, characterized by diminished ECM remodeling, prolonged chondrocyte survival, delayed vascular recruitment and defective trabecular bone formation (resulting in drastically shortened bones). These data support the hypothesis that proper ECM remodeling is the dominant rate-limiting process for programmed cell death, angiogenesis and osteoblast recruitment during normal skeletal morphogenesis.

c-Jun and JunB Antagonistically Control Cytokine-Regulated Mesenchymal–Epidermal Interaction in Skin

Interactions between mesenchymal and epithelial cells are responsible for organogenesis and tissue homeostasis. This mutual cross-talk involves cell surface proteins and soluble factors, which are mostly the result of regulated transcription. To elucidate dimer-specific functions of the AP-1 family of transcription factors, we reconstituted skin by combining primary human keratinocytes and mouse wild-type, c-jun(-/-), and junB(-/-) fibroblasts. We have discovered an antagonistic function of these AP-1 subunits in the fibroblast-mediated paracrine control of keratinocyte proliferation and differentiation, and traced this effect to the IL-1-dependent regulation of KGF and GM-CSF. These data suggest that the relative activation state of these AP-1 subunits in a non-cell-autonomous, transregulatory fashion directs regeneration of the epidermis and maintenance of tissue homeostasis in skin.

Function and regulation of AP-1 subunits in skin physiology and pathology

The mouse skin has become the model of choice to study the regulation and function of AP-1 subunits in many physiological and pathological processes in vivo and in vitro. Genetically modified mice, in vitro reconstituted skin equivalents and epidermal cell lines were established, in which AP-1-regulated genetic programs of cell proliferation, differentiation and tumorigenesis can be analysed. Since the epidermis, as our interface with the environment, is subjected to radiation and injury, signal transduction pathways and critical AP-1 members regulating the mammalian stress response could be identified. Regulated expression of important components of the cytokine network, cell surface receptors and proteases, which orchestrate the process of wound healing has been found to rely on AP-1 activity. Here we review our current knowledge on the function of AP-1 subunits and AP-1 target genes in these fascinating fields of skin physiology and pathology.

JunB is essential for mammalian placentation

Lack of JunB, an immediate early gene product and member of the AP‐1 transcription factor family causes embryonic lethality between E8.5 and E10.0. Although mutant embryos are severely retarded in growth and development, cellular proliferation is apparently not impaired. Retardation and embryonic death are caused by the inability of JunB‐deficient embryos to establish proper vascular interactions with the maternal circulation due to multiple defects in extra‐embryonic tissues. The onset of the phenotypic defects correlates well with high expression of junB in wild‐type extra‐embryonic tissues. In trophoblasts, the lack of JunB causes a deregulation of proliferin, matrix metalloproteinase‐9 (MMP‐9) and urokinase plasminogen activator (uPA) gene expression, resulting in a defective neovascularization of the decidua. As a result of downregulation of the VEGF‐receptor 1 (flt‐1), blood vessels in the yolk sac mesoderm appeared dilated. Mutant embryos which escape these initial defects finally die from a non‐vascularized placental labyrinth. Injection of junB−/− embryonic stem (ES) cells into tetraploid wild‐type blastocysts resulted in a partial rescue, in which the ES cell‐derived fetuses were no longer growth retarded and displayed a normal placental labyrinth. Therefore, JunB appears to be involved in multiple signaling pathways regulating genes involved in the establishment of a proper feto‐maternal circulatory system.

Chronic Myeloid Leukemia with Increased Granulocyte Progenitors in Mice Lacking JunB Expression in the Myeloid Lineage

The functions of JunB during myelopoiesis were studied in vivo. Transgenic mice specifically lacking JunB expression in the myeloid lineage (junB(-/-)Ubi-junB mice) develop a transplantable myeloproliferative disease eventually progressing to blast crisis, which resembles human chronic myeloid leukemia. Similarly, mice reconstituted with ES cell-derived junB-/- fetal liver cells also develop a myeloproliferative disease. In both cases, the absence of JunB expression results in increased numbers of granulocyte progenitors, which display enhanced GM-CSF-mediated proliferation and extended survival, associated with changes in the expression levels of the GM-CSFalpha receptor, the anti-apoptotic proteins Bcl2 and Bclx, and the cell cycle regulators p16(INK4a) and c-Jun. Importantly, ectopic expression of JunB fully reverts the immature and hyperproliferative phenotype of JunB-deficient myeloid cells. These results identify JunB as a key transcriptional regulator of myelopoiesis and a potential tumor suppressor gene.

Mice lacking JunB are osteopenic due to cell-autonomous osteoblast and osteoclast defects

Because JunB is an essential gene for placentation, it was conditionally deleted in the embryo proper. JunB Δ/Δ mice are born viable, but develop severe low turnover osteopenia caused by apparent cell-autonomous osteoblast and osteoclast defects before a chronic myeloid leukemia-like disease. Although JunB was reported to be a negative regulator of cell proliferation, junB Δ/Δ osteoclast precursors and osteoblasts show reduced proliferation along with a differentiation defect in vivo and in vitro. Mutant osteoblasts express elevated p16INK4a levels, but exhibit decreased cyclin D1 and cyclin A expression. Runx2 is transiently increased during osteoblast differentiation in vitro, whereas mature osteoblast markers such as osteocalcin and bone sialoprotein are strongly reduced. To support a cell-autonomous function of JunB in osteoclasts, junB was inactivated specifically in the macrophage–osteoclast lineage. Mutant mice develop an osteopetrosis-like phenotype with increased bone mass and reduced numbers of osteoclasts. Thus, these data reveal a novel function of JunB as a positive regulator controlling primarily osteoblast as well as osteoclast activity.

Both AP-1 and Cbfa1-like factors are required for the induction of interstitial collagenase by parathyroid hormone

PTH is a major regulator of calcium homeostasis by mobilizing calcium through bone resorption. We show that the expression of collagenase-3 (MMP-13), a member of the family of matrix metalloproteinases, required for the cleavage of collagens in the bone, is increased upon PTH injection in mice. A cis-acting element in the collagenase-3 promoter was identified which, together with AP-1, is required for induction by PTH. This element contains CCACA motifs which are required for binding of the 65 kDa osteoblast-specific splice variant of Cbfa1. Introduction of mutations in this binding site that interfere with protein interaction also eliminates PTH inducibilty and transactivation by Cbfa/Runt proteins. While DNA binding activity of AP-1 is increased upon PTH treatment, high basal level of Cbfa/Runt binding activity is detectable in untreated cells which is not further increased by PTH, suggesting that AP-1 and Cbfa1 contribute to transcriptional activation through different mechanisms. In agreement with the critical role of both proteins defined in tissue culture cells, expression of collagenase-3 is reduced in mice lacking c-fos and is completely absent in cbfa1−/− embryos. These data provide the first evidence for a critical role of Cbfa1, a major regulator of bone development, in PTH-dependent processes such as bone resorption.

Th2 cell-specific cytokine expression and allergen-induced airway inflammation depend on JunB

Naïve CD4+ T cells differentiate into effector T helper 1 (Th1) or Th2 cells, which are classified by their specific set of cytokines. Here we demonstrate that loss of JunB in in vitro polarized Th2 cells led to a dysregulated expression of the Th2‐specific cytokines IL‐4 and IL‐5. These cells produce IFN‐γ and express T‐bet, the key regulator of Th1 cells. In line with the essential role of Th2 cells in the pathogenesis of allergic asthma, mice with JunB‐deficient CD4+ T cells exhibited an impaired allergen‐induced airway inflammation. This study demonstrates novel functions of JunB in the development of Th2 effector cells, for a normal Th2 cytokine expression pattern and for a complete Th2‐dependent immune response in mice.

Critical role for NF-κB-induced JunB in VEGF regulation and tumor angiogenesis

Regulation of vascular endothelial growth factor (VEGF) expression is a complex process involving a plethora of transcriptional regulators. The AP‐1 transcription factor is considered as facilitator of hypoxia‐induced VEGF expression through interaction with hypoxia‐inducible factor (HIF) which plays a major role in mediating the cellular hypoxia response. As yet, both the decisive AP‐1 subunit leading to VEGF induction and the molecular mechanism by which this subunit is activated have not been deciphered. Here, we demonstrate that the AP‐1 subunit junB is a target gene of hypoxia‐induced signaling via NF‐κB. Loss of JunB in various cell types results in severely impaired hypoxia‐induced VEGF expression, although HIF is present and becomes stabilized. Thus, we identify JunB as a critical independent regulator of VEGF transcription and provide a mechanistic explanation for the inherent vascular phenotypes seen in JunB‐deficient embryos, ex vivo allantois explants and in vitro differentiated embryoid bodies. In support of these findings, tumor angiogenesis was impaired in junB−/− teratocarcinomas because of severely impaired paracrine‐acting VEGF and the subsequent inability to efficiently recruit host‐derived vessels.