Outer root sheath (ORS) cells organize into epidermoid cyst-like spheroids when cultured inside Matrigel: a light-microscopic and immunohistological comparison between human ORS cells and interfollicular keratinocytes

In organotypic cultures, outer root sheath (ORS) cells of the human hair follicle develop into a stratified epithelium largely reminiscent of the epidermis; this apparently reflects their importance during wound healing. In the present study, ORS cells were grown inside a three-dimensional network of extracellular matrix proteins (Matrigel), together with different mesenchymal cells, in an attempt to mimic their follicular environment. Thus, inside Matrigel, ORS cells formed spheroids differentiating toward the center and showing all the markers of epidermal keratinization. Under identical conditions, normal epidermal keratinocytes developed similar spheroids, but of a significantly smaller size. Human dermal fibroblasts and dermal papilla cells, cocultured in the matrix, had a positive influence on both the proliferation and differentiation within both types of spheroids. Epidermal differentiation markers, such as suprabasal keratins, involucrin, filaggrin, gp80 and pemphigoid antigen, were readily expressed in ORS spheroids, whereas hard (hair) keratins were not detectable by immunostaining. Cells positive for an epithelial membrane antigen, strongly expressed in sebaceous glands, were seen in numerous spheroids. In contrast to organotypic surface epithelia, the expression and location of different integrin chains was normalized in ORS spheroids, indicating an enhanced mesenchymal influence in this in vitro system.     

Outer root sheath (ORS) cells organize into epidermoid cyst-like spheroids when cultured inside Matrigel: a light-microscopic and immunohistological comparison between human ORS cells and interfollicular keratinocytes

In organotypic cultures, outer root sheath (ORS) cells of the human hair follicle develop into a stratified epithelium largely reminiscent of the epidermis; this apparently reflects their importance during wound healing. In the present study, ORS cells were grown inside a three-dimensional network of extracellular matrix proteins (Matrigel), together with different mesenchymal cells, in an attempt to mimic their follicular environment. Thus, inside Matrigel, ORS cells formed spheroids differentiating toward the center and showing all the markers of epidermal keratinization. Under identical conditions, normal epidermal keratinocytes developed similar spheroids, but of a significantly smaller size. Human dermal fibroblasts and dermal papilla cells, cocultured in the matrix, had a positive influence on both the proliferation and differentiation within both types of spheroids. Epidermal differentiation markers, such as suprabasal keratins, involucrin, filaggrin, gp80 and pemphigoid antigen, were readily expressed in ORS spheroids, whereas hard (hair) keratins were not detectable by immunostaining. Cells positive for an epithelial membrane antigen, strongly expressed in sebaceous glands, were seen in numerous spheroids. In contrast to organotypic “surface” epithelia, the expression and location of different integrin chains was normalized in ORS spheroids, indicating an enhanced mesenchymal influence in this in vitro system.     

Restoration of the epidermal phenotype by follicular outer root sheath cells in recombinant culture with dermal fibroblasts

In order to better understand how outer root sheath (ORS) cells are able to reepithelialize superficial skin wounds, the level of epidermal differentiation achieved by isolated ORS cells in vitro was determined. Using postmitotic human dermal fibroblasts (HDF) as efficient feeder cells, large numbers of ORS cells from individual follicles were generated. Passaged ORS cells were grown exposed to air on HDF-populated collagen gels in the CRD device (Noser and Limat, In vitro 23, 541-545, 1987) which allows histiotypic tissue organization. In such recombinant organotypic cultures, ORS cells developed distinct epidermal strata comparable to interfollicular keratinocytes (NEK). Ultrastructurally, desmosomes and intermediate filaments increased in number toward the epithelial surface and small keratohyalin (KH) granules (but no large irregular KH granules as in NEK) were abundant, adjacent to an electrondense stratum corneum. Also, synthesis of epidermal suprabasal keratins (K1 and 10;2D gels) was lower in ORS cultures, but clearly visible suprabasally by immunofluorescence along with other epidermal markers (involucrin, filaggrin, surface glycoprotein gp80, pemphigus vulgaris antigen). Basement membrane components (laminin, type IV collagen, bullous pemphigoid antigen) were detectable in both ORS and NEK in these assays. Thus, phenotypic expression was largely comparable, but, whereas terminal differentiation (keratinization) was progressing in NEK cultures limiting their lifespan, this seemed to be better controlled in ORS cultures and viable cell layers persisted resulting in longer survival time.     

Delayed wound healing in keratin 6a knockout mice

Keratin 6 (K6) expression in the epidermis has two components: constitutive expression in the innermost layer of the outer root sheath (ORS) of hair follicles and inducible expression in the interfollicular epidermis in response to stressful stimuli such as wounding. Mice express two K6 isoforms, MK6a and MK6b. To gain insight into the functional significance of these isoforms, we generated MK6a-deficient mice through mouse embryonic stem cell technology. Upon wounding, MK6a was induced in the outer ORS and the interfollicular epidermis including the basal cell layer of MK6a(+/+) mice, whereas MK6b induction in MK6a(-/-) mice was restricted to the suprabasal layers of the epidermis. After superficial wounding of the epidermis by tape stripping, MK6a(-/-) mice showed a delay in reepithelialization from the hair follicle. However, the healing of full-thickness skin wounds was not impaired in MK6a(-/-) animals. Migration and proliferation of MK6a(-/-) keratinocytes were not impaired in vitro. Furthermore, the migrating and the proliferating keratinocytes of full-thickness wounds in MK6a(-/-) animals expressed neither MK6a nor MK6b. These data indicate that MK6a does not play a major role in keratinocyte proliferation or migration but point to a role in the activation of follicular keratinocytes after wounding. This study represents the first report of a keratin null mutation that results in a wound healing defect.     

Outer root sheath cells of human hair follicle are able to regenerate a fully differentiated epidermis in vitro

During wound healing, interfollicular epidermis can be regenerated from the outer root sheath of hair follicles, showing that the cells of this structure can shift toward an interfollicular epidermal phenotype. Similarly, it has been shown that a multilayered epithelium originating from outer sheath cells can be obtained in vitro by culturing hair follicles. However, in the culture systems developed so far, the phenotypical shift was incomplete since the cells retained some of their original characteristics and did not acquire several key markers of terminally differentiated epidermis. In this paper, we describe a new tissue culture method for obtaining a multilayered epithelium from outer sheath cells. This is performed by implanting human hair follicles vertically into dermal equivalents and then raising the culture at the air-liquid interface. The morphological, immunological, and biochemical features of the in vitro reconstructed tissue are very similar to those observed in normal interfollicular epidermis, including those specific for terminally differentiated keratinocytes. Thus, under appropriate in vitro conditions, outer root sheath cells are able to express an interfollicular epidermal phenotype as occurs in vivo during wound healing.     

Differential expression of stem cell markers in human follicular bulge and interfollicular epidermal compartments

Although skin contains a number of stem cell repositories, their characterization has been hindered by a lack of specific markers and an unclear in vivo localization. In this study, we whole mounted single human scalp hair follicles and examined their profiles using in situ immunohistochemistry and multicolor immunofluorescence in search of markers to distinguish between stem cells residing in the interfollicular epidermis (IFE) and bulge. Our study revealed that expression of several biomarkers localized uniquely to the basal IFE (CD34 and CD117), bulge region (CD200), or both (CK15, CD49f, and CD29). In addition, we found that both basal IFE and bulge stem cells did not express CD71 or CD24 suggesting their potential utility as negative selection markers. Dermal papilla but not basal IFE or bulge stem cells expressed CD90, making it a potential positive selection marker for dermal hair follicle stem cells. The markers tested in this study may enable pursuit of cell sorting and purification strategies aimed at determining each stem cell population’s unique molecular signature.     

常见毛囊细胞角蛋白在毛囊周期中的表达研究

目的探讨常见毛囊细胞角蛋白在毛囊周期中的表达特征。 方法取毛囊发育期、生长期启动、生长期、退化期和静止期的小鼠皮肤,石蜡切片后通过免疫荧光的方法,检测细胞角蛋白Krt5、Krt6、Krt10、Krt14、Krt15和Krt19的表达情况。 结果Krt5在静止期和生长期启动表达于所有毛囊上皮细胞,在其他时期表达不一致;Krt6表达于所有时期的外根鞘细胞和内根鞘细胞;Krt10表达于生长期和退化期的毛母质和内根鞘细胞,在其他时期表达不一致;Krt14在生长期和退化期表达于所有毛囊上皮细胞,在其他时期表达不一致;Krt15和Krt19表达于毛囊发育期、生长期启动和静止期的毛囊隆突区细胞,在生长期和退化期表达不一致。 结论角蛋白作为毛囊结构或毛囊干细胞标记物仅适用于特定的毛囊周期。研究者在使用毛囊角蛋白作为标记物时,应首先明确其在毛囊周期中的表达情况。     

Acidic and basic hair/nail ("hard") keratins: their colocalization in upper cortical and cuticle cells of the human hair follicle and their relationship to "soft" keratins

Although numerous hair proteins have been studied biochemically and many have been sequenced, relatively little is known about their in situ distribution and differential expression in the hair follicle. To study this problem, we have prepared several mouse monoclonal antibodies that recognize different classes of human hair proteins. Our AE14 antibody recognizes a group of 10-25K hair proteins which most likely corresponds to the high sulfur proteins, our AE12 and AE13 antibodies define a doublet of 44K/46K proteins which are relatively acidic and correspond to the type I low sulfur keratins, and our previously described AE3 antibody recognizes a triplet of 56K/59K/60K proteins which are relatively basic and correspond to the type II low sulfur keratins. Using these and other immunological probes, we demonstrate the following. The acidic 44K/46K and basic 56-60K hair keratins appear coordinately in upper corticle and cuticle cells. The 10-25K, AE14-reactive antigens are expressed only later in more matured corticle cells that are in the upper elongation zone, but these antigens are absent from cuticle cells. The 10-nm filaments of the inner root sheath cells fail to react with any of our monoclonal antibodies and are therefore immunologically distinguishable from the cortex and cuticle filaments. Nail plate contains 10-20% soft keratins in addition to large amounts of hair keratins; these soft keratins have been identified as the 50K/58K and 48K/56K keratin pairs. Taken together, these results suggest that the precursor cells of hair cortex and nail plate share a major pathway of epithelial differentiation, and that the acidic 44K/46K and basic 56-60K hard keratins represent a co- expressed keratin pair which can serve as a marker for hair/nail-type epithelial differentiation.     

Expression of keratin K14 in the epidermis and hair follicle: insights into complex programs of differentiation

Keratins K14 and K5 have long been considered to be biochemical markers of the stratified squamous epithelia, including epidermis (Moll, R., W. Franke, D. Schiller, B. Geiger, and R. Krepler. 1982. Cell. 31:11-24; Nelson, W., and T.-T. Sun. 1983. J. Cell Biol. 97:244-251). When cells of most stratified squamous epithelia differentiate, they downregulate expression of mRNAs encoding these two keratins and induce expression of new sets of keratins specific for individual programs of epithelial differentiation. Frequently, as in the case of epidermis, the expression of differentiation-specific keratins also leads to a reorganization of the keratin filament network, including denser bundling of the keratin fibers. We report here the use of monospecific antisera and cRNA probes to examine the differential expression of keratin K14 in the complex tissue of human skin. Using in situ hybridizations and immunoelectron microscopy, we find that the patterns of K14 expression and filament organization in the hair follicle are strikingly different from epidermis. Some of the mitotically active outer root sheath (ORS) cells, which give rise to ORS under normal circumstances and to epidermis during wound healing, produce only low levels of K14. These cells have fewer keratin filaments than basal epidermal cells, and the filaments are organized into looser, more delicate bundles than is typical for epidermis. As these cells differentiate, they elevate their expression of K14 and produce denser bundles of keratin filaments more typical of epidermis. In contrast to basal cells of epidermis and ORS, matrix cells, which are relatively undifferentiated and which can give rise to inner root sheath, cuticle and hair shaft, show no evidence of K14, K14 mRNA expression, or keratin filament formation. As matrix cells differentiate, they produce hair-specific keratins and dense bundles of keratin filaments but they do not induce K14 expression. Collectively, the patterns of K14 and K14 mRNA expression and filament organization in mitotically active epithelial cells of the skin correlate with their relative degree of pluripotency, and this suggests a possible basis for the deviation of hair follicle programs of differentiation from those of other stratified squamous epithelia.     

The catalog of human hair keratins. I. Expression of the nine type I members in the hair follicle.

The human type I hair keratin subfamily comprises nine individual members, which can be subdivided into three groups. Group A (hHa1, hHa3-I, hHa3-II, hHa4) and B (hHa7, hHa8) each contains structurally related hair keratins, whereas group C members hHa2, hHa5, and hHa6 represent structurally rather unrelated hair keratins. Antibodies produced against these individual hair keratins, first analyzed for specificity by one- dimensional Western blots of total hair keratins, were used to establish the two-dimensional catalog of the human type I hair keratin subfamily. The catalog comprises two different series of type I hair keratins: a strongly expressed, Coomassie-stainable series containing hair keratins hHa1, hHa3-I/II, hHa4, and hHa5, and a weakly expressed, immunodetectable series harboring hHa2, hHa6 hHa7, and hHa8. In situ hybridization and immunohistochemical expression studies on scalp follicles show that two hair keratins, hHa2 and hHa5, define the early stage of hair differentiation, i.e. hHa5 expression in hair matrix and hHa5/hHa2 coexpression in the early hair cuticle cells. Whereas cuticular differentiation proceeds without the expression of further type I hair keratins, matrix cells embark on the cortical pathway by sequentially expressing hHa1, hHa3-I/II, and hHa4, which are supplemented by hHa6 at an advanced stage of cortical differentiation, and hHa8, which is expressed heterogeneously in cortex cells. Thus, six type I hair keratins are involved in the terminal differentiation of anagen hairs. The expression of hHa7 is conspicuously different from that of the other hair keratins in that it does not occur in the large anagen follicles of terminal scalp hairs but only in central cortex cells of the rare and small follicle type that gives rise to vellus hairs.     

Expression of hair-related keratins in a soft epithelium: subpopulations of human and mouse dorsal tongue keratinocytes express keratin markers for hair-, skin- and esophageal-types of differentiation

The dorsal surfaces of mammalian tongues are covered with numerous projections known as filiform papillae whose morphology varies in different species. Using a panel of monoclonal antibodies to keratins as probes, we have established that, in both human and mouse, the interpapillary epithelia express mainly the "esophageal-type" keratins, while the papillary epithelia express "skin-type" keratins as well as some keratins reacting with a monoclonal antibody (AE13) to hair keratins. The AE13-reactive proteins of the mouse were found to be very similar to those of authentic mouse hair keratins. However, the corresponding protein of human tongue appears to be different from all known human keratins. This protein has a MW of 51K; it is relatively acidic; it is sulfhydryl-rich, as revealed by iodoacetic acid-induced charge and apparent size shift; it shares an epitope with all the known acidic human hair keratins; and it is associated with keratin fibrils in vivo. This protein may therefore be regarded as a novel type I "hard" keratin. These data establish that mammalian dorsal tongue epithelia can be divided into at least three compartments that undergo mainly "esophageal-", "skin-" and "hair"-types of differentiation. Different keratin filaments, e.g., those of the esophageal- and hair-types, exhibit strikingly different degrees of lateral aggregation, which can potentially account for the different physical strength and rigidity of various cellular compartments. Our data also suggest the possibility that variations in papillary structure in human and mouse may arise from different spatial arrangements of specific keratinocytes, and/or from the expression of specialized hair-related keratins.     

Spatial and temporal control of laminin-332 (5) and -511 (10) expression during induction of anagen hair growth.

Basement membrane plays important roles in hair growth. We characterized changes in laminin isoform expression during hair cycling. At the mRNA level, laminin-511 (10) expression underwent a steady increase during anagen stages. In contrast, laminin-332 (5) expression was initially upregulated in outer root sheath (ORS) keratinocytes at anagen II and then transiently downregulated. Laminin-332 significantly increased coincident with the signal in inner root sheath and hair matrix cells after anagen IV. Levels of laminin-332 proteins were also upregulated at late anagen I-III but dropped after anagen IV. This decrease coincided with increased levels of mRNA encoding the two proteases, membrane type 1 metalloproteinase and bone morphogenetic protein 1, involved in laminin-332 processing. Immunohistochemistry demonstrated that laminin-332 and alpha6 beta4 integrin were well colocalized, but their signals were remarkably decreased in the lower half of follicles after anagen VI. Consistent with these data, ultrastructurally mature hemidesmosomes were seen in ORS keratinocytes at anagen II, whereas at anagen VI, only fragmental hemidesmosomes were present. In hair follicle culture, laminin-511 (10)/521 (11)-rich human placental laminin enhanced hair growth, whereas recombinant laminin-332 antagonized hair growth induced by laminin-511. Our results indicate a positive role for laminin-511 and a negative role for laminin-332 on hair growth.     

Expression of epidermal keratins and filaggrin during human fetal skin development

The major structural proteins of epithelia, the keratins, and the keratin filament-associated protein, filaggrin, were analyzed in more than 50 samples of human embryonic and fetal skin by one-dimensional SDS PAGE and immunoblotting with monoclonal and polyclonal antibodies. Companion samples were examined by immunohistochemistry and electron microscopy. Based on structural characteristics of the epidermis, four periods of human epidermal development were identified. The first is the embryonic period (before 9 wk estimated gestational age), and the others are within the fetal period: stratification (9-14 wk), follicular keratinization (14-24 wk), and interfollicular keratinization (beginning at approximately 24 wk). Keratin proteins of both the acidic (AE1-reactive, type I) and the basic (AE3-reactive, type II) subfamilies were present throughout development. Keratin intermediate filaments were recognized in the tissue by electron microscopy and immunohistochemical staining. Keratins of 50 and 58 kD were present in the epidermis at all ages studied (8 wk to birth), and those of 56.5 and 67 kD were expressed at the time of stratification and increased in abundance as development proceeded. 40- and 52-kD keratins were present early in development but disappeared with keratinization. Immunohistochemical staining suggested the presence of keratins of 50 and 58 kD in basal cells, 56.5 and 67 kD in intermediate cells, and 40 and 52 kD in the periderm as well as in the basal cells between the time of stratification and birth. Filaggrin was first detected biochemically at approximately 15 wk and was localized immunohistochemically in the keratinizing cells that surround hair follicles. It was identified 8-10 wk later in the granular and cornified cell layers of keratinized interfollicular epidermis. These results demonstrate the following. An intimate relationship exists between expression of structural proteins and morphologic changes during development of the epidermis. The order of expression of individual keratins is consistent with the known expression of keratins in simple vs. stratified vs. keratinized epithelia. Expression of keratins typical of stratified epithelia (50 and 58 kD) precedes stratification, and expression of keratins typical of keratinization (56.5 and 67 kD) precedes keratinization, which suggests that their expression marks the tissue commitment to those processes. Because only keratins that have been demonstrated in various adult tissues are expressed during fetal development, we conclude that there are no "fetal" keratins per se.(ABSTRACT TRUNCATED AT 400 WORDS)     

Perspectives on hair evolution based on some comparative studies on vertebrate cornification

Hair evolution contributed to the biological success of mammals. Hair origin from synapsid scales is speculative and requires extensive modifications of the morphogenetic process transforming lens-shaped dermis of scales into small dermal papillae in hair. Hair evolution from glands is hypothetical but is supported from studies on the signaling control of hair vs. glandular morphogenesis. Based on immunocytochemical and comparative studies, it is hypothesized that the onion-like organization of hair derived from glandular pegs which central part produced lipids and some keratin. In a following stage, involucrin, trichohyalin, and keratins were produced in the central cells of the gland and formed a solid medulla surrounded by keratinocytes of the inner root sheath. The origin of this protohair was possibly related to increased concentration of beta-catenin and other signaling molecules in epithelial cells following the evolution of a dermal papilla. The latter activated the keratogenic genes, already utilized in cells of the claws, in concentric layers of cells of the glandular peg. Lipidogenic genes were depressed. As new genes evolved in the genome of synapsids, new circular layers of keratinocytes containing specialized hard keratins and keratin-associated proteins were formed around medullary cells. The new keratinocytes probably originated the cortex separating medulla from the external cells that became the inner root sheath. The hypothesis indicates that in a following stage, the medulla was obliterated or replaced by cortical cells while the external part of the cortex formed a cuticular surface due to the different growth rate with inner root sheath cells.     

Urokinase plasminogen activator (uPA) is a positive regulator of outer root sheath keratinocyte proliferation

Urokinase plasminogen activator (uPA), a serine proteinase, is important in the development and epidermal wound healing, and seems to play a regulatory role in the proliferation of mouse epidermal keratinocytes (KC). In the present study, we found detectable uPA expression in outer root sheath (ORS) KC in the early anagen phase in mouse vibrissa follicles, but not in the late anagen or in the telogen and categen phases. uPA was also detected in ORS KC cultured from neonatal mice vibrissa. Specific exogenous inhibitors of uPA, amiloride and uPA antibody, significantly reduced the proliferation of ORS KC. Thus uPA is consistently elevated in the hyperproliferative hair follicle KC, and inhibition of the enzyme decreases hair follicle KC proliferation. We deduce that uPA is a very important mediator of the hair follicle cycle because its activity correlates with ORS KC proliferation.     

Ultrastructural localization of hair keratin homologs in the claw of the lizard Anolis carolinensis

The claw of lizards is largely composed of beta‐keratins, also referred to as keratin‐associated beta‐proteins. Recently, we have reported that the genome of the lizard Anolis carolinensis contains alpha keratin genes homologous to hair keratins typical of hairs and claws of mammals. Molecular and immunohistochemical studies demonstrated that two hair keratin homologs named hard acid keratin 1 (HA1) and hard basic keratin 1 (HB1) are expressed in keratinocytes forming the claws of A. carolinensis. Here, we extended the immunocytochemical localization of the novel reptilian keratins to the ultrastructural level. After sectioning, claws were subjected to immunogold labeling using antibodies against HA1, HB1, and, for comparison, beta‐keratins. Electron microscopy showed that the randomly organized network of tonofilaments in basal and suprabasal keratinocytes becomes organized in long and parallel bundles of keratin in precorneous layers, resembling cortical cells of hairs. Entering the cornified part of the claw, the elongated corneous cells fuse and accumulate corneous material. HA1 and HB1 are absent in the basal layer and lower spinosus layers of the claw and are expressed in the upper and precorneous layers, including the elongating corneocytes. The labeling for alpha‐keratin was loosely associated with filament structures forming the fibrous framework of the claws. The ultrastructural distribution pattern of hard alpha‐keratins resembled that of beta‐keratins, which is compatible with the hypothesis of an interaction during claw morphogenesis. The data on the ultrastructural localization of hair keratin homologs facilitate a comparison of lizard claws and mammalian hard epidermal appendages containing hair keratins. J. Morphol., 2011. © 2010 Wiley‐Liss, Inc.     

Expression of galectin-1 and -3 and of accessible binding sites during murine hair cycle

Although protein-carbohydrate interactions are supposed to play key roles in cell adhesion, signalling and growth control. Their exact role in skin physiology has only recently been investigated. The endogenous lectins galectin-1 and galectin-3 have been identified in skin including hair follicles. Here, we analyzed the expression and distribution of these galectins and their binding sites in C57BL/6 mice during hair cycle. The expression of galectin-1 and galectin-3 binding sites was found to be predominantly hair cycle-dependent showing some overlapping to the expression of galectin-1 and -3. The outer root sheath (ORS) expressed galectin-1 binding sites during anagen IV to VI and in early catagen, whereas galectin-1 was expressed from early anagen to late catagen. The ORS expressed galectin-3 binding sites during catagen transition corresponding to a galectin-3 expression during anagen V and catagen. The innermost layer of the ORS expressed galectin-3 binding sites during anagen VI until catagen VIII, but galectin-3 during anagen III to IV and catagen. The inner root sheath (IRS) expressed galectin-3 binding sites only in anagen IV but missed expression of any of the two galectins. The matrix cells expressed galectin-3 binding sites in catagen II-III as well as galectin-3 during anagen V to catagen IV. The present study provides the first evidence for a cycle-related expression of both galectin-1 and -3 and their binding sites during murine hair cycle.     

Involucrin, but not filaggrin and Kdap mRNA, expression is downregulated in 3-D cultures of intact rat hair bulbs after calcium stimulation

The hair follicle consists of several distinctive epidermal cell layers. The hair root, which undergoes keratinization, is surrounded by two sheaths: the inner root sheath (IRS) and the outer root sheath (ORS). The ORS is continuous with the basal layer of the epidermis. Its cells do not keratinize in situ, unlike IRS. We have previously demonstrated that keratinization of the ORS was prevented by contact with the IRS in hair follicle mid-segments (i.e. fragments dissected from skin at the level above the hair bulb and below the opening of the sebaceous gland duct) cultured on agarose layer. The purpose of this study was to determine whether the same applies to the hair bulb. After isolation, intact bulbs or hair bulb-derived cells were incubated in suspension in a low or high calcium medium. The level of mRNA for differentiation markers: involucrin, filaggrin, keratinocyte differentiation associated protein and trichohyalin, was studied by RealTime PCR. We observed increased Ca(2+) upregulated expression of involucrin, filaggrin, trichohyalin and Kdap in cultures of bulb-derived cells, but in hair bulbs downregulation of involucrin and trichohyalin was observed. We concluded that the inner root sheath exerts an inhibitory effect on the expression of involucrin and trichohyalin already in the hair bulbs. The observation that downregulation of involucrin expression under Ca(2+) influence occurs both in hair bulb and midsegments could simplify future experiments, since their separation does not seem to be necessary.