Robert M. Lavker

Existence of slow-cycling limbal epithelial basal cells that can be preferentially stimulated to proliferate: Implications on epithelial stem cells

Despite the obvious importance of epithelial stem cells in tissue homeostasis and tumorigenesis, little is known about their specific location or biological characteristics. Using 3H-thymidine labeling, we have identified a subpopulation of corneal epithelial basal cells, located in the peripheral cornea in a region called limbus, that are normally slow cycling, but can be stimulated to proliferate in response to wounding and to a tumor promotor, TPA. No such cells can be detected in the central corneal epithelium, suggesting that corneal epithelial stem cells are located in the limbus. A comparison of various types of epithelial stem cells revealed a common set of features, including their preferred location, pigment protection, and growth properties, which presumably play a crucial role in epithelial stem cell function.

Involvement of Follicular Stem Cells in Forming Not Only the Follicle but Also the Epidermis

The location of follicular and epidermal stem cells in mammalian skin is a crucial issue in cutaneous biology. We demonstrate that hair follicular stem cells, located in the bulge region, can give rise to several cell types of the hair follicle as well as upper follicular cells. Moreover, we devised a double-label technique to show that upper follicular keratinocytes emigrate into the epidermis in normal newborn mouse skin, and in adult mouse skin in response to a penetrating wound. These findings indicate that the hair follicle represents a major repository of keratinocyte stem cells in mouse skin, and that follicular bulge stem cells are potentially bipotent as they can give rise to not only the hair follicle, but also the epidermis.

Cumulative effects from repeated exposures to suberythemal doses of UVB and UVA in human skin

BACKGROUND The skin is repeatedly exposed to solar UV radiation. Long-term photodamage is a consequence of cumulative UV radiation injury. Hence an examination of the repetitive effects of UV exposure is more likely to yield clues to the early alterations that lead to photoaged skin than a single exposure. OBJECTIVE We examined the effects of repetitive low-dose UV irradiation on human skin with the aim of identifying UVA-induced effects that may have a different wavelength dependence than acute erythema. METHODS Areas on the lower part of the back were each exposed to a suberythemal dose (0.5 minimal erythema dose [MED]) of solar simulated radiation (290 to 400 nm) and of UVA (320 to 400 nm) once daily, 5 days a week, for 28 doses. One site was also treated daily with a sunscreen having a sun protection factor of 22 and then exposed to 11 MEDs of solar simulated radiation for the same duration. Epidermal and dermal changes were analyzed and quantified by histochemical stains in combination with computer-assisted image analysis of tissue sections. RESULTS At equal 0.5 MED doses, UVA induced greater cumulative changes than solar simulated radiation, as assessed by development of a greater cumulative erythema response in the first week of treatment, the presence of epidermal hyperplasia and stratum corneum thickening, depletion of Langerhans cells, dermal inflammatory infiltrates, and deposition of lysozyme on elastin fibers. These changes were not prevented by the sunscreen. A single short-term dose of UVA did not elicit these changes. CONCLUSION These findings suggest that UVA may contribute significantly to long-term actinic damage and that the spectral dependence for cumulative damage does not parallel the action spectrum for acute injury (erythema) in human beings.

Ocular surface epithelia contain ABCG2-dependent side population cells exhibiting features associated with stem cells

When cell populations are incubated with the DNA-binding dye Hoechst 33342 and subjected to flow cytometry analysis for Hoechst 33342 emissions, active efflux of the dye by the ABCG2/BCRP1 transporter causes certain cells to appear as a segregated cohort, known as a side population (SP). Stem cells from several tissues have been shown to possess the SP phenotype. As the lack of specific surface markers has hindered the isolation and subsequent biochemical characterization of epithelial stem cells this study sought to determine the existence of SP cells and expression of ABCG2 in the epithelia of the ocular surface and evaluate whether such SP cells had features associated with epithelial stem cells. Human and rabbit limbal-corneal and conjunctival epithelial cells were incubated with Hoechst 33342, and analyzed and sorted by flow cytometry. Sorted cells were subjected to several tests to determine whether the isolated SP cells displayed features consistent with the stem cell phenotype. Side populations amounting to <1% of total cells, which were sensitive to the ABCG2-inhibitor fumitremorgin C, were found in the conjunctival and limbal epithelia, but were absent from the stem cell-free corneal epithelium. Immunohistochemistry was used to establish the spatial expression pattern of ABCG2. The antigen was detected in clusters of conjunctival and limbal epithelia basal cells but was not present in the corneal epithelium. SP cells were characterized by extremely low light side scattering and contained a high percentage of cells that: showed slow cycling prior to tissue collection; exhibited an initial delay in proliferation after culturing; and displayed clonogenic capacity and resistance to phorbol-induced differentiation; all features that are consistent with a stem cell phenotype.

MicroRNA-184 antagonizes microRNA-205 to maintain SHIP2 levels in epithelia.

Despite their potential to regulate approximately one-third of the whole genome, relatively few microRNA (miRNA) targets have been experimentally validated, particularly in stratified squamous epithelia. Here we demonstrate not only that the lipid phosphatase SHIP2 is a target of miRNA-205 (miR-205) in epithelial cells, but, more importantly, that the corneal epithelial-specific miR-184 can interfere with the ability of miR-205 to suppress SHIP2 levels. This is the first example of a miRNA negatively regulating another to maintain levels of a target protein. Interfering with miR-205 function by using a synthetic antagomir, or by the ectopic expression of miR-184, leads to a coordinated damping of the Akt signaling pathway via SHIP2 induction. This was associated with a marked increase in keratinocyte apoptosis and cell death. Aggressive squamous cell carcinoma (SCC) cells exhibited elevated levels of miR-205. This was associated with a concomitant reduction in SHIP2 levels. Partial knockdown of endogenous miR-205 in SCCs markedly decreased phosphorylated Akt and phosphorylated BAD levels and increased apoptosis. We were able to increase SHIP2 levels in SCC cells after inhibition of miR-205. Therefore, miR-205 might have diagnostic value in determining the aggressivity of SCCs. Blockage of miR-205 activity with an antagomir or via ectopic expression of miR-184 could be novel therapeutic approaches for treating aggressive SCCs.

The anatomy and pathogenesis of wrinkles

The anatomy of linear wrinkles (‘crows feet’ and temporal frown lines), fine criss‐cross wrinkles of the face and wrinkling of the general body surface of elderly persons, was studied by light and scanning electron microscopy. No histological features distinguished the various wrinkles from surrounding skin. It was concluded that the wrinkle is a configurational change, like the grooves worn into an old glove, without specific structural alterations at the histological level. As regards pathogenesis, the common setting was found to be deterioration of the elastic tissue network. The skin becomes looser, excessive, and loses the ability to snap back to its original state after being deformed.

Reaction of mast cell proteases tryptase and chymase with protease activated receptors (PARs) on keratinocytes and fibroblasts.

Protease activated receptors (PARs) compose a family of G protein signal transduction receptors activated by proteolysis. In this study, the susceptibility of PARs expressed on human keratinocytes and dermal fibroblasts to the human mast cell proteases tryptase and chymase was evaluated. PAR activation was measured by monitoring cytosolic [Ca2+] in cells loaded with the fluorescent Ca2+ probe Fura‐2. Tryptase produced transient cytosolic Ca2+ mobilization in keratinocytes, but not in fibroblasts. Ca2+ mobilization in keratinocytes required enzymatically active tryptase, demonstrated desensitization, and was blocked by pretreatment of cells with the PAR‐2 peptide agonist SLIGKV, trypsin, or the phospholipase inhibitor U73122. Heparin, a GAG that binds to tryptase, stabilizing its functional form, also inhibited tryptase‐induced Ca2+ mobilization. The maximal response elicited by tryptase was smaller than that observed upon treatment of keratinocytes with trypsin, a known activator of PAR‐2, and keratinocytes made refractory to tryptase by pretreatment with the protease remained responsive to trypsin. Pretreatment of keratinocytes with thrombin, an activator of PAR‐1 and ‐3 (thrombin receptors), had no detectable effect on the tryptase or trypsin responses. These data suggest that in keratinocytes tryptase may be activating a subpopulation of PAR‐2 receptors. Treatment of keratinocytes or fibroblasts with human chymase did not produce Ca2+ mobilization, nor did it affect Ca2+ mobilization produced by trypsin. However, chymase pretreatment of fibroblasts rapidly inhibited the ability of these cells to respond to thrombin. Inhibition was dependent on chymase enzymatic activity and was not significantly affected by the presence of heparin. This finding is consistent with studies indicating that PAR‐1 may be susceptible to proteases with chymotrypsin‐like specificity. These results suggest that the proteases tryptase and chymase secreted from mast cells in skin may affect the behavior of surrounding cells by the hydrolysis of PARs expressed by these cells. J. Cell. Physiol. 176:365–373, 1998.

Mitochondrial reactive oxygen species promote epidermal differentiation and hair follicle development

Skin development requires reactive oxygen species generated by mitochondria in keratinocytes. Building a Barrier Mitochondria are an important source of reactive oxygen species (ROS), which participate in diverse signaling pathways. To test the role of mitochondrially produced ROS in epidermal development, Hamanaka et al. generated mice with keratinocytes lacking mitochondrial transcription factor A (TFAM), which is required for transcription of genes encoded by mitochondrial DNA, including those that proteins required for ROS generation. The epidermis of these mice was abnormally thick, lacked hair, and showed defects in differentiation and barrier function, which likely contributed to perinatal death. Keratinocytes from these mice did not produce mitochondrial ROS and showed impaired Notch signaling, which is involved in epidermal differentiation, and β-catenin signaling, which is required for growth of hair follicles. Thus, signaling pathways involved in skin development rely on the production of ROS generated by mitochondria. Proper regulation of keratinocyte differentiation within the epidermis and follicular epithelium is essential for maintenance of epidermal barrier function and hair growth. The signaling intermediates that regulate the morphological and genetic changes associated with epidermal and follicular differentiation remain poorly understood. We tested the hypothesis that reactive oxygen species (ROS) generated by mitochondria are an important regulator of epidermal differentiation by generating mice with a keratinocyte-specific deficiency in mitochondrial transcription factor A (TFAM), which is required for the transcription of mitochondrial genes encoding electron transport chain subunits. Ablation of TFAM in keratinocytes impaired epidermal differentiation and hair follicle growth and resulted in death 2 weeks after birth. TFAM-deficient keratinocytes failed to generate mitochondria-derived ROS, a deficiency that prevented the transmission of Notch and β-catenin signals essential for epidermal differentiation and hair follicle development, respectively. In vitro keratinocyte differentiation was inhibited in the presence of antioxidants, and the decreased differentiation marker abundance in TFAM-deficient keratinocytes was partly rescued by application of exogenous hydrogen peroxide. These findings indicate that mitochondria-generated ROS are critical mediators of cellular differentiation and tissue morphogenesis.

Ultrastructure of the human vomeronasal organ.

Virtually all vertebrates have a vomeronasal system whose involvement in pheromone detection plays a crucial role in reproduction. In humans, the vomeronasal organ has been assumed to be vestigial or absent and without functional significance. In the present study involving over 400 subjects, vomeronasal pits were observed in all individuals except those with pathological conditions affecting the septum. Electron microscopy of the adult human vomeronasal organ indicates the presence of two potential receptor elements in the pseudostratified epithelial lining: microvillar cells, and unmyelinated, intraepithelial axons. In addition, unmyelinated axons are common in the lamina propria surrounding the organ. They appear to constitute the components essential for a functional chemosensory system, and may thus provide the basis for a pheromone detection system as in other animals.

Anatomy of striae

The histopathology of striae distensae is disputed; different authorities give contradictory accounts of the microscopic changes, especially in elastic fibres.