Shared and independent functions of aPKCλ and Par3 in skin tumorigenesis.

The polarity proteins Par3 and aPKC are key regulators of processes altered in cancer. Par3/aPKC are thought to dynamically interact with Par6 but increasing evidence suggests that aPKC and Par3 also exert complex-independent functions. Whereas aPKCλ serves as tumor promotor, Par3 can either promote or suppress tumorigenesis. Here we asked whether and how Par3 and aPKCλ genetically interact to control two-stage skin carcinogenesis. Epidermal loss of Par3, aPKCλ, or both, strongly reduced tumor multiplicity and increased latency but inhibited invasion to similar extents, indicating that Par3 and aPKCλ function as a complex to promote tumorigenesis. Molecularly, Par3/aPKCλ cooperate to promote Akt, ERK and NF-κB signaling during tumor initiation to sustain growth, whereas aPKCλ dominates in promoting survival. In the inflammatory tumorigenesis phase Par3/aPKCλ cooperate to drive Stat3 activation and hyperproliferation. Unexpectedly, the reduced inflammatory signaling did not alter carcinogen-induced immune cell numbers but reduced IL-4 Receptor-positive stromal macrophage numbers in all mutant mice, suggesting that epidermal aPKCλ and Par3 promote a tumor-permissive environment. Importantly, aPKCλ also serves a distinct, carcinogen-independent role in controlling skin immune cell homeostasis. Collectively, our data demonstrates that Par3 and aPKCλ cooperate to promote skin tumor initiation and progression, likely through sustaining growth, survival, and inflammatory signaling.

Ceramide synthase 4 regulates stem cell homeostasis and hair follicle cycling.

Ceramides are crucial for skin barrier function, but little is known about the regulation of epidermal appendages and whether stem cell populations that control their regeneration depend on specific ceramide species. Here we demonstrate that ceramide synthase 4 (CerS4) is highly expressed in the epidermis of adult mice where it is localized in the interfollicular epidermis and defined populations within the pilosebaceous unit. Inactivation of CerS4 in mice resulted in precocious activation of hair follicle bulge stem cells while expanding the Lrig1(+) junctional zone and sebaceous glands. This was preceded first by a decrease in bone morphogenetic protein (BMP) and a subsequent increase in Wnt signaling. This imbalance in quiescent versus activating signals likely promoted a prolonged anagen-like hair follicle state after the second catagen, which exhausted stem cells over time ultimately resulting in hair loss in aged mice. K14-Cre-mediated deletion of CerS4 revealed a similar phenotype, thus suggesting an epidermis intrinsic function of CerS4 in regulating the regeneration of the pilosebaceous unit. The data indicate that CerS4-directed epidermal ceramide composition is essential to control hair follicle stem and progenitor cell behavior potentially through its regulation of BMP and Wnt signaling.

Epidermal polarity genes in health and disease.

The epidermis of the skin is a highly polarized, metabolic tissue with important innate immune functions. The polarity of the epidermis is, for example, reflected in controlled changes in cell shape that accompany differentiation, oriented cell division, and the planar orientation of hair follicles and cilia. The establishment and maintenance of polarity is organized by a diverse set of polarity proteins that include transmembrane adhesion proteins, cytoskeletal scaffold proteins, and kinases. Although polarity proteins have been extensively studied in cell culture and in vivo in simple epithelia of lower organisms, their role in mammalian tissue biology is only slowly evolving. This article will address the importance of polarizing processes and their molecular regulators in epidermal morphogenesis and homeostasis and discuss how alterations in polarity may contribute to skin disease.

Mammalian aPKC/Par polarity complex mediated regulation of epithelial division orientation and cell fate.

Oriented cell division is a key regulator of tissue architecture and crucial for morphogenesis and homeostasis. Balanced regulation of proliferation and differentiation is an essential property of tissues not only to drive morphogenesis but also to maintain and restore homeostasis. In many tissues orientation of cell division is coupled to the regulation of differentiation producing daughters with similar (symmetric cell division, SCD) or differential fate (asymmetric cell division, ACD). This allows the organism to generate cell lineage diversity from a small pool of stem and progenitor cells. Division orientation and/or the ratio of ACD/SCD need to be tightly controlled. Loss of orientation or an altered ratio can promote overgrowth, alter tissue architecture and induce aberrant differentiation, and have been linked to morphogenetic diseases, cancer and aging. A key requirement for oriented division is the presence of a polarity axis, which can be established through cell intrinsic and/or extrinsic signals. Polarity proteins translate such internal and external cues to drive polarization. In this review we will focus on the role of the polarity complex aPKC/Par3/Par6 in the regulation of division orientation and cell fate in different mammalian epithelia. We will compare the conserved function of this complex in mitotic spindle orientation and distribution of cell fate determinants and highlight common and differential mechanisms in which this complex is used by tissues to adapt division orientation and cell fate to the specific properties of the epithelium.

aPKCλ controls epidermal homeostasis and stem cell fate through regulation of division orientation.

The atypical protein kinase C (aPKC) is a key regulator of polarity and cell fate in lower organisms. However, whether mammalian aPKCs control stem cells and fate in vivo is not known. Here we show that loss of aPKCλ in a self-renewing epithelium, the epidermis, disturbed tissue homeostasis, differentiation, and stem cell dynamics, causing progressive changes in this tissue. This was accompanied by a gradual loss of quiescent hair follicle bulge stem cells and a temporary increase in proliferating progenitors. Lineage tracing analysis showed that loss of aPKCλ altered the fate of lower bulge/hair germ stem cells. This ultimately led to loss of proliferative potential, stem cell exhaustion, alopecia, and premature aging. Inactivation of aPKCλ produced more asymmetric divisions in different compartments, including the bulge. Thus, aPKCλ is crucial for homeostasis of self-renewing stratifying epithelia, and for the regulation of cell fate, differentiation, and maintenance of epidermal bulge stem cells likely through its role in balancing symmetric and asymmetric division.

Development of a three-dimensional in vitro model for longitudinal observation of cell behavior: monitoring by magnetic resonance imaging and optical imaging.

PURPOSE: The aim of this study is the development of a three-dimensional multicellular spheroid cell culture model for the longitudinal comparative and large-scale screening of cancer cell proliferation with noninvasive molecular imaging techniques under controlled and quantifiable conditions. PROCEDURES: The human glioblastoma cell line Gli36DeltaEGFR was genetically modified to constitutively express the fluorescence protein mCherry, and additionally labeled with iron oxide nanoparticles for high-field MRI detection. The proliferation of aggregates was longitudinally monitored with fluorescence imaging and correlated with aggregate size by light microscopy, while MRI measurements served localization in 3D space. Irradiation with gamma-rays was used to detect proliferational response. RESULTS: Cell proliferation in the stationary three-dimensional model can be observed over days with high accuracy. A linear relationship of fluorescence intensity with cell aggregate size was found, allowing absolute quantitation of cells in a wide range of cell amounts. Glioblastoma cells showed pronounced suppression of proliferation for several days following high-dose gamma-irradiation. CONCLUSIONS: Through the combination of two-dimensional optical imaging and 3D MRI, the position of individual cell aggregates and their corresponding light emission can be detected. This allows an exact quantification of cell proliferation, with a focus on very small cell amounts (below 100 cells) using high resolution noninvasive techniques as a well-controlled basis for further cell transplantation studies.