Multiple mechanisms of dissociated epidermal cell spreading.

To test the possibility that epidermal cells use a common basement membrane protein whenever they spread, in vitro experiments were conducted using trypsin-dissociated guinea pig epidermal cells and the following proteins: human serum, bovine serum albumin, serum fibronectin, Type IV collagen, laminin, and epibolin (a recently described serum glycoprotein which supports epidermal cell spreading; Stenn, K.S., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:6907.). When the cells were added to media containing the specific proteins, all the tested proteins, except for serum albumin, supported cell spreading. Added to protein-coated substrates in defined media, the cells spread on fibronectin, epibolin, and laminin-Type IV collagen, but not on albumin or whole serum. In none of these experiments were the results qualitatively affected by the presence of cycloheximide. Antibodies to a specific protein blocked cell spreading on that protein but not on the other active proteins, e.g. whereas antibodies to epibolin blocked cell spreading on epibolin, they did not affect spreading on fibronectin, collagen, or laminin. In a second assay in which the cells were allowed to adhere to tissue culture plastic before the protein-containing medium was added, the cells spread only if the medium contained epibolin. Moreover, under these conditions the spreading activity of whole serum and plasma was neutralized by antiepibolin antibodies. These results support the conclusion that dissociated epidermal cells possess multiple spreading modes which depend, in part, on the proteins of the substrate, proteins of the medium, and the sequence of cell adhesion and protein exposure.

Coepibolin, the activity of human serum that enhances the cell spreading properties of epibolin, associates with albumin.

Earlier studies have shown that epibolin, a glycoprotein in human plasma, identical to preparations from other laboratories referred to as serum spreading factor and vitronectin, requires a second plasma activity in order to support maximal primary epidermal cell spreading. This plasma fraction, referred to as coepibolin, alone supports no cell spreading but potentiates the activity of epibolin by at least 5-10 times. Studies reported here indicate that coepibolin activity associates with one plasma fraction, it cannot be substituted by commercial preparations of ovalbumin, soybean trypsin inhibitor, or hemoglobin, but it can be substituted by commercial bovine or human serum albumin. The activity purified to electrophoretic homogeneity comigrates with, and shows antigenic properties of albumin. Under the assay conditions coepibolin affects the extent and morphology of cell spreading: in the presence of epibolin plus coepibolin cells assume a more polar orientation than in epibolin alone. For its maximal effect coepibolin must be present continuously in the early phases of cell spreading.

Organ culture of adult mouse esophageal mucosa in a defined medium.

A method is described for culturing adult mouse esophageal mucosa in a chemically defined medium. The preparation consists of mucosa and superficial submucosa. By light microscopy, autoradiography, and tritiated-thymidine [3H]TdR) uptake the tissue appears viable for at least 3 days. Although a drop in the rate of [3H]TdR uptake is observed in the initial hours of culture, recovery occurs by 24 hr and uptake remains constant for at least an additional 48 hr. Twenty-four-hour exposure to [3H]TdR and autoradiography reveals that 93 +/- 3% of the basal cells take up label; appreciable labeling is not found in other cells of the preparation. Pulse labeling indicated a transit time from basal layer to keratin layer of about 72 hr. This preparation should be useful for short-term in vitro studies of keratinized, stratified, squamous epithelium free of appendages and for studies of the growth properties of esophageal mucosa under simulated pathologic conditions.

Fibronectin (LETS) does not support epithelial cell spreading.

The dependence of epithelial cell spreading on fibronectin-containing media has been investigated, using purified human plasma fibronectin, and two in vitro assays: (1) epidermal outgrowths from measured pieces of mouse ear skin, and (2) spreading of dissociated cells from trypsinized guinea pig epidermis. Using these systems, appreciable spread occurred in media containing whole plasma and plasma deficient in fibronectin but no significant spread occurred in media enriched only in fibronectin. Returning fibronectin to deficient plasma did not enhance or reduce optimal cell spreading. These studies suggest that the factor(s) in human plasma which supports epithelial cell spreading in vitro is not fibronectin and that the chemistry of epithelial and fibroblast cell spreading is different.

Epidermal cell confluence and implications for a two-step mechanism of wound closure.

In contrast to freshly isolated cells, some cultured keratinocytes have the ability to adhere and spread in protein-free media. Reported here are experiments testing the hypothesis that the social history of keratinocytes influences their ability to spread in defined media. The experiments indicate that confluent cells lack the ability to spread in defined media while subconfluent cells have this property. The inability of dissociated confluent cells to spread in protein-free media is referred to phenomenologically as a "confluent block." The confluent block is acquired rapidly (1-3 days) and lost slowly (5-7 days). The ability of subconfluent cells to spread in the absence of media protein is sensitive to cycloheximide. Aortic endothelial cells and dermal fibroblasts do not demonstrate a confluent block. These observations are consonant with a two-step mechanism of epidermal wound repair: the first occurs immediately after wounding during which the cells require substratum-active proteins, and the second occurs 5-7 days later when the cells are able to synthesize their own substratum.

Phorbol ester serves as a coepibolin in the spreading of primary guinea pig epidermal cells.

In earlier studies, it was shown that the human plasma-spreading glycoprotein, epibolin (the 65 kD species of serum-spreading factor or vitronectin), requires a second plasma component, termed coepibolin, in order to support maximal dissociated epidermal cell spreading in tissue culture. Whereas epibolin alone in defined medium supports some cell spreading, the purified plasma coepibolin preparations do not effect spreading in the absence of epibolin. Although not yet entirely purified, coepibolin associates with some plasma fractions but not with others; it is certainly not a property of all proteins, e.g., while bovine serum albumin (BSA) has coepibolin activity, ovalbumin does not. The data presented here show that the phorbol ester, 12-tetra-decanoyl-1-phorbol-13-acetate (TPA) can act as a potent coepibolin and support maximal spreading over a concentration range of 10-100 ng/ml. In the absence of epibolin TPA does not stimulate the spreading of epidermal cells when given alone or in the presence of BSA or ovalbumin. Coepibolin activity appears to associate with tumor-promoting activity in that the phorbol derivative, phorbol-12,13-didecanoate, shows coepibolin activity, while its inactive non-tumor-promoting isomer, phorbol-4 alpha-phorbol-12,13-didecanoate, does not. These data suggest that the proteinaceous plasma-derived cofactor acts in a fashion similar to TPA and that this as yet unexplained mechanism of TPA action is important to the full expression of epibolin and to the early phase of epidermal cell spreading.

Changes in expression of apoptosis-associated genes in skin mark early catagen.

Programmed cell death is central to hair biology, as the hair follicle undergoes cycles of growth (anagen), regression (catagen), and rest (telogen). During catagen, the hair follicle shortens via a pathway of programmed cell death and apoptosis. The molecular mechanisms involved in this process have not been elucidated yet. Using reverse transcriptase-polymerase chain reaction, we examined in this study the expression in total skin, throughout one hair cycle, of a series of regulatory genes associated with apoptosis. We show that gene expression within skin is hair-cycle-dependent. Transforming growth factor-beta was expressed immediately before catagen; therefore, it might be involved in the early signaling of this process. Tumor necrosis factor-beta was expressed during catagen and might be involved in follicular apoptosis. Several proto-oncogenes and transcription factors have been described in the regulation of apoptosis in other systems. Here we show that the transcript levels of c-myc, c-myb, and c-jun changed immediately before or during early catagen and thus could be involved in the signaling or regulation of catagen. Levels of p53 remained constant throughout anagen and catagen, suggesting that p53 is not involved in the developmentally induced apoptosis of the hair follicle. The variable expression throughout the hair cycle of the genes described demonstrates the dynamic changes of the skin and underscores the importance of studying the complete hair cycle when characterizing any molecule in skin.

The angiogenic properties of the rat vibrissa hair follicle associate with the bulb.

As the hair follicle is one of the most rapidly growing tissues in the body, it must be nourished by a rich blood supply. Histological studies have indicated that the number of vessels about a growing follicle exceeds that about a resting follicle, so we postulated that the hair follicle might provide its own angiogenic stimulus during certain phases of its growth. Reported here are experiments testing the angiogenic properties of the growing (anagen) hair follicle. Using the rabbit corneal pocket angiogenesis assay and cycled anagen rat vibrissae hair follicles, we found that the mesenchymal dermal papilla had no angiogenic properties, but the anagen bulb was angiogenic. These findings suggest a mechanism for the cycling of hair follicles and an example of normal epithelium to mesenchyme interactions.

Expression of the bcl-2 protooncogene in the cycling adult mouse hair follicle.

The hair follicle undergoes a cycle of growing, regressing, and resting phases (anagen, catagen, telogen, respectively). As the follicle enters catagen, the cells of the lower, cycling portion undergo a process of controlled cell death (apoptosis). Understanding the mechanism of apoptosis in the follicle should give insight into one of the control steps of hair cycling. In this study we sought the expression of bcl-2, a protooncogene associated with apoptosis control, in the cycling follicle of the adult mouse. Using a monoclonal antibody to the mouse protein we immunolocalized bcl-2 gene product in the cycling pelage follicle of the C57/B6 adult mouse. The protein was expressed in the follicular papilla (a non-cycling portion of the follicle) throughout the cycle-including telogen. The cycling follicular epithelium, however, showed positive antibody staining in anagen, which decreased in catagen and disappeared in telogen. In anagen the cells of the bulb, bulge, and basal layer of the outer root sheath expressed the bcl-2 protein. Understanding the action of this apoptosis-inhibiting molecule should serve to elucidate the dynamics of follicular cycling.

Dispase, a neutral protease from Bacillus polymyxa, is a powerful fibronectinase and type IV collagenase.

Dispase, a neutral protease isolated from culture filtrates of Bacillus polymyxa, has proven to be a rapid, effective, but gentle agent for separating intact epidermis from the dermis and intact epithelial sheets in culture from the substratum. In both cases it effects separation by cleaving the basement membrane zone region while preserving the viability of the epithelial cells. Because it is not known what or where in the basement membrane zone Dispase cleaves, we set up studies to define its substrate specificity. Using purified basement membrane components and sodium dodecyl sulfate-polyacrylamide gel electrophoresis we show that Dispase cleaves fibronectin and type IV collagen, but not laminin, type V collagen, serum albumin, or transferrin. The action of Dispase on collagen appears to be selective for type IV collagen in that several stable degradation products are formed, whereas the enzyme degrades type I collagen only minimally. In newborn human skin, as seen by electron microscopy, Dispase removes the lamina densa, rich in type IV collagen, but preserves the anchoring fibrils (structures known to contain type VII collagen) and the epidermal cells. Because its action is so selective, it suggests that Dispase can serve as a powerful tool for dissecting epithelial-mesenchymal interactions.

Vitronectin: effects on keratinocyte motility and inhibition of collagen-induced motility.

Epibolin, a plasma protein, was initially purified on the basis of its ability to enhance spreading of keratinocytes. It is now known that epibolin is identical to serum spreading factor, S protein, and vitronectin, and the current name for the protein is vitronectin. Studies of vitronectin on cultured keratinocytes showed that it caused spreading and epiboly but not cellular adhesion to the substratum. In studies with other types of cells, vitronectin increased migration of several types of cells in a Boyden chamber. Because some agents that enhance spreading and adhesion, such as collagen and fibronectin, also increase motility, we tested whether vitronectin increased motility of keratinocytes. By photographing and quantitating motility of keratinocytes plated on a bed of colloidal gold particles, we determined that vitronectin increased local movement of keratinocytes in a concentration-dependent fashion, resulting in clearing of gold particles in a circular pattern around the cells, but did not cause the production of tracks found in cultures plated on collagen or fibronectin. The small increases in clearing of the gold particles that occurred in the presence of vitronectin were abolished by antibody to vitronectin. Furthermore, the marked increase in motility produced by type I collagen was significantly reduced when the keratinocytes were treated with vitronectin. Antibody to vitronectin also abrogated the vitronectin-induced reduction in collagen-stimulated motility, confirming that this action was specific for vitronectin. Serum, which contains vitronectin, stimulated motility in a fashion identical to purified vitronectin, but serum lacking vitronectin was inactive. These studies show that vitronectin causes a localized increase in movement associated with spreading resulting in a halo around individual cells, that vitronectin does not enhance directional motility of keratinocytes in this assay but in contrast antagonizes such motility produced by collagen, and that vitronectin is the factor in serum responsible for this effect. The findings with vitronectin and collagen show that these agents stimulate different types of motility. The roles in wound healing of agents stimulating different types of motility are unclear and require further study.

Epithelial growth by rat vibrissae follicles in vitro requires mesenchymal contact via native extracellular matrix.

An in vitro assay utilizing the rat vibrissa anagen follicle as a model for studying the epithelial-mesenchymal interactions (EMI) in hair growth is described. Through selective disruption of the epithelial-mesenchymal interface, we investigate whether the specialized extracellular matrix (ECM) of the dermal papilla and basement membrane zone (BMZ) serves a crucial function in hair follicle EMI. Epithelial bulbs incubated intact within their follicular sheaths incorporate thymidine primarily into cells of the hair matrix and outer root sheath, as shown by autoradiography. However, after removal of its mesenchymal associations (dermal papilla and extrabulbar connective tissue), the epithelial bulb showed no incorporation. Neither externally added collagen (type I or IV) nor the basement membrane components in Matrigel could substitute for the growth supporting influence of native surrounding stroma. Mechanical separation of the bulb from the dermal papilla in the basement membrane zone inhibited thymidine incorporation by the epithelium even though mesenchyme was still in close proximity. Enzymatic digestion of the dermal papilla ECM and the basal lamina by Dispase, a fibronectinase and type IV collagenase, also inhibited bulb growth without evidence of cytotoxicity. These experiments suggest that direct epithelial to mesenchymal contact is required for the support of follicular epithelial growth in vitro and that specific ECM components, possibly fibronectin and/or type IV collagen, rather than diffusable factors alone, play a crucial role in the mechanism of hair follicle EMI. The in vitro system described here provides an alternative to developmental EMI models and may serve as a valuable tool for studying EMI in the adult mammalian organism.

Expression of two Ig family adhesion molecules in the murine hair cycle: DCC in the bulge epithelia and NCAM in the follicular papilla.

The hair cycle involves remodeling of cells and of cell groups into a complex follicular structure. During skin appendage development, adhesion molecules such as neural cell adhesion molecule (NCAM) and deleted in colon carcinoma (DC) participate in the formation of cell groups. NCAM has been found to be expressed in the mesenchyme during mouse hair follicle induction. DCC expression has been observed in the epithelial cells of the developing feather. We postulate that these two molecules may also define cell groups in the cycling hair follicle. Here we report their spatio-temporal expression patterns during the depilation-induced murine hair cycle. NCAM expression was also examined in positive and negative hair-inductive follicular papilla cell lines. Throughout the hair cycle, DCC expression was confined to the basal keratinocytes of the epidermis and the epithelial portion of the hair follicle. During mid-anagen, two types of deleted in colon carcinoma staining were observed. One was a cell surface pattern seen in the epithelial cells in the bulge region where the follicular stem cells reside. The other was a diffuse cytoplasmic staining pattern in the transient hair follicle epithelia located below the bulge region. Prominent NCAM staining was observed in the follicular papilla throughout the hair cycle and was accompanied by weak staining of the matrix epithelia. NCAM expression correlated with hair induction by a follicular papilla cell line. The results suggest that DCC and NCAM define the permanent cell groups of the hair follicle and that NCAM is important for hair induction.

A comprehensive guide for the accurate classification of murine hair follicles in distinct hair cycle stages.

Numerous strains of mice with defined mutations display pronounced abnormalities of hair follicle cycling, even in the absence of overt alterations of the skin and hair phenotype; however, in order to recognize even subtle, hair cycle-related abnormalities, it is critically important to be able to determine accurately and classify the major stages of the normal murine hair cycle. In this comprehensive guide, we present pragmatic basic and auxiliary criteria for recognizing key stages of hair follicle growth (anagen), regression (catagen) and quiescence (telogen) in C57BL/6NCrlBR mice, which are largely based on previous work from other authors. For each stage, a schematic drawing and representative micrographs are provided in order to illustrate these criteria. The basic criteria can be employed for all mouse strains and require only routine histochemical techniques. The auxiliary criteria depend on the immunohistochemical analysis of three markers (interleukin-1 receptor type I, transforming growth factor-beta receptor type II, and neural cell-adhesion molecule), which allow a refined analysis of anatomical hair follicle compartments during all hair cycle stages. In contrast to prior staging systems, we suggest dividing anagen III into three distinct substages, based on morphologic differences, onset and progression of melanogenesis, and the position of the dermal papilla in the subcutis. The computer-generated schematic representations of each stage are presented with the aim of standardizing reports on follicular gene and protein expression patterns. This guide should become a useful tool when screening new mouse mutants or mice treated with pharmaceuticals for discrete morphologic abnormalities of hair follicle cycling in a highly reproducible, easily applicable, and quantifiable manner.

Molecules of the cycling hair follicle--a tabulated review.

In this review we tabulated molecules which have been experimentally identified to be associated with, or play a role in, hair follicle growth. While compiling these data we were impressed by the fact that this field is only now beginning to be developed in terms of molecular analysis. Ironically, hair was used in some of the earliest molecular approaches to biologic structure (e.g. Astbury and Street, 1931), but the field did not develop from there. From our review we have come to the following conclusions. (1) As indicated by the growing number of reports dealing with follicle-associated molecules in the past 3 years, the field of hair biology has entered a new molecular era. (2) In many reported hair biology studies not enough emphasis has been placed on the fact that the follicle is a dynamic structure. All too often a study is limited to follicles of one particular phase of the cycle or one phase of development. Students in the field have to be more sensitive to the remarkable changes that this deceptively simple structure can undergo during its cycle. (3) Although we have not been able to find any molecules unique to the follicle, some of the structural molecules come close to an ideal tool. It is our impression that even more specific molecule tags will be found. Whether this requires a subtraction library approach or gene mapping of specific mutants is not yet clear. It would appear that the large, diverse family of intermediate filament-associated proteins will prove to be an excellent source of unique follicle-labeling molecules. (4) There is an acute need for molecules which distinguish the phases of the cycle, e.g. telogen from early anagen. Telogen is by far the most difficult phase to identify morphologically since the earliest phase of anagen and the latest phase of catagen may appear structurally like telogen. That these phases are functionally distinguishable must imply a molecular difference. As the number of recognized hair follicle-associated molecules and their interactions increase, it will be essential to assemble libraries of highly specific RNA and antibody probes for localization and mapping studies. We recognize that this review, as written, is imperfect. It is particularly deficient in making any effort towards identifying unifying principles of structure and function. We look forward to returning to this subject within 3 years.(ABSTRACT TRUNCATED AT 400 WORDS)

Calcinosis cutis following extravasation of calcium chloride.

A 27-year-old woman with hypoparathyroidism developed multiple firm white-yellow papules along the path of an infiltrated calcium chloride intravenous infusion. A biopsy specimen obtained ten days after the extravasation revealed an urticarial reaction. A subsequent biopsy specimen, obtained 25 days after the extravasation, showed diffuse dermal calcification, confirmed by roentgenographic analysis, with incipient transepidermal elimination. A biopsy specimen obtained 40 days after the extravasation was consistent with an elimination reaction. An increase in mast cells was not noted. Electron microscopy showed mineral deposits along collagen fibrils without significant collagenous degeneration. In this report we describe a complication of intravenous calcium chloride infusion.

Metastasizing atypical fibroxanthoma. Coexistence with chronic lymphocytic leukemia.

Atypical fibroxanthoma (AFX) is one of a group of cutaneous lesions with a malignant histological appearance but a generally benign clinical course. A 79-year-old white man had AFX of the cheek that recurred and metastasized to buccal and cervical lymph nodes three months after initial diagnosis. When careful physical and and laboratory examinations were done, the patient was found to have concomitant chronic lymphatic leukemia, "null cell" type. In view of the low incidence of metastasizing AFX and the increased occurrence of tumors in patients with lymphomatous disorders, an important association is suggested. Before establishing the prognosis for patients with pseudomalignancies of the skin, an evaluation of their general health and immunological status should be made.

Human serum and epithelial spread in tissue culture.

The dependence of epithelial spread on human serum in culture has been studied. Using measured pieces of mouse ear skin epithelial outgrowth about floating explants (epiboly) and from adherent explants was studied. Where compared directly the two systems show similar results. Because of its ease of quantitation, the adherent explant culture was studied in greater detail. In this system in the presence of serum appreciable spread was found only after 48 h but spread continued for at least the next 4 days. In the absence of serum only minimal epithelial spread occurred. Adding serum to deficient media enhanced spreading and removing serum from media depressed spreading. The extent of spread appeared independent of mouse age for the first 4-10 weeks though 2-week-old mouse skin showed quantitatively greater spreading. The activity in human serum responsible for epithelial spread acts under conditions of minimal DNA synthesis and is not reproduced by bovine serum albumin, fetuin, or serum that had been exposed to 100 degrees C for 5 min. The activity is not dialyzable and it is resistant to the protease inhibitors DFP and PMSF. These studies suggest that a specific serum component(s) serves to support epithelial spread in vitro.

Hair follicle growth controls.

Research in hair biology has embarked in the pursuit for molecules that control hair growth. Many molecules already have been associated with the controls of hair patterning, hair maturation, and hair cycling and differentiation. Knowing how these molecules work gives us the tools for understanding and treating patients with hair disorders.