The in vivo function of mammalian cell and tissue polarity regulators--how to shape and maintain the epidermal barrier.

The establishment and maintenance of cell and tissue polarity is crucial for a range of biological processes, such as oriented division, migration, adhesion and barrier function. The molecular pathways that regulate cell and tissue polarity have been extensively studied in lower organisms as well as in mammalian cell culture. By contrast, relatively little is still known about how polarization regulates the in vivo formation and homeostasis of mammalian tissues. Several recent papers have identified crucial roles for mammalian polarity proteins in a range of in vivo processes, including stem cell behavior, cell fate determination, junction formation and maintenance and organ development. Using the epidermis of the skin as a model system, this Commentary aims to discuss the in vivo significance of cell and tissue polarity in the regulation of mammalian tissue morphogenesis, homeostasis and disease. Specifically, we discuss the mechanisms by which the molecular players previously identified to determine polarity in vitro and/or in lower organisms regulate epidermal stratification; orient cell division to drive cell fate determination within the epidermal lineage; and orient hair follicles. We also describe how altered polarity signaling contributes to skin cancer.

The disintegrin/metalloproteinase Adam10 is essential for epidermal integrity and Notch-mediated signaling.

The disintegrin and metalloproteinase Adam10 has been implicated in the regulation of key signaling pathways that determine skin morphogenesis and homeostasis. To address the in vivo relevance of Adam10 in the epidermis, we have selectively disrupted Adam10 during skin morphogenesis and in adult skin. K14-Cre driven epidermal Adam10 deletion leads to perinatal lethality, barrier impairment and absence of sebaceous glands. A reduction of spinous layers, not associated with differences in either proliferation or apoptosis, indicates that loss of Adam10 triggers a premature differentiation of spinous keratinocytes. The few surviving K14-Adam10-deleted mice and mice in which Adam10 was deleted postnatally showed loss of hair, malformed vibrissae, epidermal hyperproliferation, cyst formation, thymic atrophy and upregulation of the cytokine thymic stromal lymphopoetin (TSLP), thus indicating non cell-autonomous multi-organ disease resulting from a compromised barrier. Together, these phenotypes closely resemble skin specific Notch pathway loss-of-function phenotypes. Notch processing is indeed strongly reduced resulting in decreased levels of Notch intracellular domain fragment and functional Notch signaling. The data identify Adam10 as the major Site-2 processing enzyme for Notch in the epidermis in vivo, and thus as a central regulator of skin development and maintenance.

Analysis of bulge stem cells from the epidermis using flow cytometry.

The epidermis is a multilayered epithelium consisting of multiple different progenitor cell populations, all of which are important to epidermal function. In order to study these populations, several techniques have been developed that enable specific purification of the different progenitor cell populations. The best characterized stem cell population in the epidermis, and likely the most pluripotent, are the quiescent stem cells in the hair follicle bulge. In this chapter, we provide a method for isolating bulge stem cells from skin of adult mice using fluorescence-activated cell sorting of immunofluorescently labeled keratinocytes. We use the cell surface markers CD34 and α6-integrin for the enrichment of bulge stem cells. This method also contains notes on how to adjust the cytometer settings for a reproducible analysis.

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.

Ubiquitin-dependent proteolytic control of SUMO conjugates.

Posttranslational protein modification with small ubiquitin-related modifier (SUMO) is an important regulatory mechanism implicated in many cellular processes, including several of biomedical relevance. We report that inhibition of the proteasome leads to accumulation of proteins that are simultaneously conjugated to both SUMO and ubiquitin in yeast and in human cells. A similar accumulation of such conjugates was detected in Saccharomyces cerevisiae ubc4 ubc5 cells as well as in mutants lacking two RING finger proteins, Ris1 and Hex3/Slx5-Slx8, that bind to SUMO as well as to the ubiquitin-conjugating enzyme Ubc4. In vitro, Hex3-Slx8 complexes promote Ubc4-dependent ubiquitylation. Together these data identify a previously unrecognized pathway that mediates the proteolytic down-regulation of sumoylated proteins. Formation of substrate-linked SUMO chains promotes targeting of SUMO-modified substrates for ubiquitin-mediated proteolysis. Genetic and biochemical evidence indicates that SUMO conjugation can ultimately lead to inactivation of sumoylated substrates by polysumoylation and/or ubiquitin-dependent degradation. Simultaneous inhibition of both mechanisms leads to severe phenotypic defects.

Alteration of organic acid metabolism in Arabidopsis overexpressing the maize C4 NADP-malic enzyme causes accelerated senescence during extended darkness.

The full-length cDNA encoding the maize (Zea mays) C(4) NADP-malic enzyme was expressed in Arabidopsis (Arabidopsis thaliana) under the control of the cauliflower mosaic virus 35S promoter. Homozygous transgenic plants (MEm) were isolated with activities ranging from 6- to 33-fold of those found in the wild type. The transformants did not show any differences in morphology and development when grown in long days; however, dark-induced senescence progressed more rapidly in MEm plants compared to the wild type. Interestingly, senescence could be retarded in the transgenic lines by exogenously supplying glucose, sucrose, or malate, suggesting that the lack of a readily mobilized carbon source is likely to be the initial factor leading to the premature induction of senescence in MEm plants. A comprehensive metabolic profiling on whole rosettes allowed determination of approximately 80 metabolites during a diurnal cycle as well as following dark-induced senescence and during metabolic complementation assays. MEm plants showed no differences in the accumulation and degradation of carbohydrates with respect to the wild type in all conditions tested, but accumulated lower levels of intermediates used as respiratory substrates, prominently malate and fumarate. The data indicated that extremely low levels of malate and fumarate are responsible for the accelerated dark-induced senescence encountered in MEm plants. Thus, in prolonged darkness these metabolites are consumed faster than in the wild type and, as a consequence, MEm plants enter irreversible senescence more rapidly. In addition, the data revealed that both malate and fumarate are important forms of fixed carbon that can be rapidly metabolized under stress conditions in Arabidopsis.