Modulation of SPARC Expression during Butyrate-Induced Terminal Differentiation of Cultured Human Keratinocytes: Regulation via a TGF-β-Dependent Pathway

Expression of SPARC (secreted protein acidic and rich in cysteine), a 43-kDa extracellular matrix-associated glycoprotein involved in tissue remodeling, was quantitated during normal human keratinocyte (NHK) growth in culture and as a function of sodium n-butyrate (NaB)-induced differentiation to mature enucleate cornified envelopes (CEs). Low levels of SPARC expression were observed in the basal-like cells of control NHKs, with isolated cells showing intense SPARC expression on the ventral surface. After addition of NaB, SPARC expression increased and the pattern of expression shifted to one involving predominantly suprabasal cells (i.e., spinous cells, pre-CEs, and mature CEs). Dense deposits of SPARC often surrounded the mature CEs. Flow cytometric analysis indicated that approximately 13% of NHKs expressed SPARC within 24 h of seeding into culture. This fraction of SPARC⁺ cells increased with time and peaked immediately postconfluence (31.3 ± 6.3% SPARC⁺). Cellular SPARC expression then decreased to baseline levels during entrance into plateau phase growth. SPARC was detectable in all phases of the cell cycle. SPARC levels were more intense and heterogeneous within the G₂/M and G₁ phases while S phase cells exhibited relatively homogeneous, low intensity, SPARC expression. During NaB-induced NHK differentiation, SPARC intracellular content increased prior to the onset of CE formation (i.e., 2 days after its addition) followed by a period of extracellular accumulation which coincided with the time of maximal CE generation (i.e., Days 4 and 5 after NaB addition). Correlation of cell size with anti-SPARC immunoreactivity revealed a predominance of SPARC expression in cells with a suprabasal phenotype. NHKs cultured on fibronectin (FN), an established modulator of epidermal cell maturation in vitro, showed a similar response to NaB. In general, however, the level of NaB-induced SPARC expression was considerably reduced in FN cultures correlating with a lower efficiency of CE formation. Induced SPARC expression was, in large part, dependent on autocrine transforming growth factor-β (TGF-β) production since incubation in the presence of NaB + neutralizing antibodies to TGF-β inhibited both the expression of SPARC by 72% and development of mature CEs.     

Cell shape changes during transition of basal keratinocytes to mature enucleate-cornified envelopes: modulation of terminal differentiation by fibronectin.

Normal human keratinocytes (NHK) in submerged culture were used to assess mechanisms associated with in vitro exposure to known stimulators (sodium butyrate; NaB) and inhibitors (fibronectin; FN) of NHK maturation. A multiparameter approach was used to define cell types generated under a range of growth conditions. Differentiation induced in response to NaB occurred through a series of morphologically distinct phenotypes and culminated in the formation of enucleate-cornified envelopes. Two-dimensional electrophoresis provided a limited database to evaluate global changes in cellular protein composition as a function of induced differentiation. Proteins were identified that characterized predominantly basal cell cultures, NaB-treated cells, and fully differentiated NHKs. Growth of NHKs on FN suppressed both spontaneous and NaB-directed maturation and inhibited maximal expression of protein changes associated with the differentiated state. Thus, the composition of the extracellular matrix can modulate (at both the morphologic and protein levels) the response of basal NHKs to a potent differentiation-inducing agent. Abrogation of NHK maturation by FN was not due to adverse effects on cellular metabolism, abortive differentiation, or altered timing of induced differentiation. FN appears to exert its suppressive effect by either maintaining an early stem cell phenotype which is poorly competent for terminal maturation or attenuating an as yet unknown aspect of the NaB-initiated differentiation cascade.     

Regulation of transglutaminase type II by transforming growth factor-beta 1 in normal and transformed human epidermal keratinocytes

This study examines the effect of transforming growth factor-beta 1 (TGF-beta 1) on the expression of Type I and II transglutaminase in normal human epidermal keratinocytes (NHEK cells). Treatment of undifferentiated NHEK cells with 100 pM TGF-beta 1 caused a 10- to 15-fold increase in the activity of a soluble transglutaminase. Based on its cellular distribution and immunoreactivity this transglutaminase was identified as Type II (tissue) transglutaminase. TGF-beta 1 did not enhance the levels of the membrane-bound Type I (epidermal) transglutaminase activity which is induced during squamous cell differentiation and did not increase Type II transglutaminase activity in differentiated NHEK cells. Several SV40 large T antigen-immortalized NHEK cell lines also exhibited a dramatic increase in transglutaminase Type II activity after TGF-beta 1 treatment; however, TGF-beta 1 did not induce any significant change in transglutaminase activity in the carcinoma-derived cell lines SCC-13, SCC-15, and SQCC/Y1. Half-maximal stimulation of transglutaminase Type II activity in NHEK cells occurred at a dose of 15 pM TGF-beta 1. TGF-beta 2 was about equally effective. This enhancement in transglutaminase activity was related to an increase in the amount of transglutaminase Type II protein as indicated by immunoblot analysis. Northern blot analyses using a specific cDNA probe for Type II transglutaminase showed that exposure of NHEK cells to TGF-beta 1 caused a marked increase in the mRNA levels of this enzyme which could be observed as early as 4 h after the addition of TGF-beta 1. Maximal induction of transglutaminase Type II mRNA occurred between 18 and 24 h. The increase in Type II transglutaminase mRNA levels was blocked by the presence of cycloheximide, suggesting that this increase in mRNA by TGF-beta 1 is dependent on protein synthesis.     

TGF-beta and retinoic acid: regulators of growth and modifiers of differentiation in human epidermal cells.

In the epidermis of skin, a fine balance exists between proliferating progenitor cells and terminally differentiating cells. We examined the effects of TGF-beta s and retinoic acid (RA) on controlling this balance in normal and malignant human epidermal keratinocytes cultured under conditions where most morphological and biochemical features of epidermis in vivo are retained. Our results revealed marked and pleiotropic effects of both TGF-beta and RA on keratinocytes. In contrast to retinoids, TGF-beta s acted on mitotically active basal cells to retard cell proliferation. Although withdrawal from the cell cycle is a necessary prerequisite for commitment to terminal differentiation, TGF-beta s inhibited normal keratinization in suprabasal cells and promoted the type of differentiation commonly associated with wound-healing and epidermal hyperproliferation. The actions of TGF-beta s and RA on normal keratinization were synergistic, whereas those on abnormal differentiation associated with hyperproliferation were antagonistic. These observations underscore the notion that environmental changes can act separately on proliferating and differentiating cells within the population. Under the conditions used here, the action of TGF-beta s on human keratinocytes was dominant over RA, and TGF-beta s did not seem to be induced as a consequence of RA treatment. This finding is consistent with the fact that RA accelerated, rather than inhibited, proliferation in raft cultures. Collectively, our data suggest that the effects of both factors on epidermal growth and differentiation are multifaceted and the extent to which their action is coupled in keratinocytes may vary under different conditions and/or in different species.     

Retinoic acid improves epidermal morphogenesis

Hyper- and hypovitaminosis A both provoke epithelial pathologies in animals and humans. This suggests that a critical level of retinoic acid (RA) is required in vivo for the maintenance of normal architecture and function of these tissues. However, no beneficial, but only adverse effects of RA on epithelia have been so far observed in vitro. For instance, addition of RA to keratinocyte cultures has been shown to inhibit epidermal differentiation while this process is stimulated by serum delipidization, which reduces RA concentration in the medium. Assuming that the previous failure to demonstrate beneficial effects of RA on the epidermal phenotype in vitro was due to culture conditions too far from the in vivo conditions we decided to reevaluate the effect of RA in a culture system optimized for epidermal morphogenesis: the "emerged dermal equivalent." When human keratinocytes were grown in such a system with total fetal calf serum, the resulting epithelium was very similar to normal epidermis. But when delipidized serum was used, the epithelium was abnormal in the direction of excessive maturation (hyperkeratosis). When physiological concentrations of RA (10(-9) and 10(-8) M) were added to the delipidized serum supplement, a normal architecture (orthokeratosis) was restored. However, as classically described in the literature, higher RA concentrations (greater than 10(-7) M) reduced epidermal maturation and produced parakeratosis. Thus, although it is unquestionable that RA reduces the synthesis of epidermal-specific differentiation markers, an optimal epidermal morphogenesis seems to be achieved only in the presence of a critical RA concentration.     

F9 Cells Can be Differentiated toward Two Distinct, Mutually Exclusive Pathways by Retinoic Acid and Sodium Butyrate

Both retinoic acid (RA) and sodium butyrate (NaB) induce differentiation in embryonal carcinoma F9 cells. Phenotypic changes caused by RA are irreversible, whereas those of NaB are rapid and reversible. In this study, we investigated the effects of combinations of these two agents on F9 cell differentiation and showed that RA had no effect on the cells induced to differentiate with NaB and vice versa. Thus, F9 cells are induced to differentiate along two distinct pathways which are mutually exclusive.     

Tgm2/Gh, Gbx1 and TGF-beta are involved in retinoic acid-induced transdifferentiation from epidermis to mucosal epithelium

We previously demonstrated that retinoic acid (RA) induces epidermis to transdifferentiate to mucosal epithelium with goblet cells in chick embryonic cultured skin. To characterize the molecular mechanism of this transdifferentiation process, we used rat embryonic cultured skin and immunohistochemistry to confirm that RA-induced epidermal transdifferentiation accompanies the expression of markers of esophagus epithelium. Because Gbx1, TG2/Gh (transglutaminase2) and TGF-beta2 are reported individually to be induced by RA in cultures of chick embryonic skin, mouse epidermal cells and human hair follicles respectively, here, we investigated whether cooperative interplay of Gbx1, TG2/Gh and TGF-beta2 is required for the transdifferentiation of epidermal cells to mucosal cells. We have shown that expression of Gbx1, TG2/Gh and TGF-beta proteins were all upregulated in RA-induced transdifferentiated skin and that the former two were expressed in the epidermis, while TGF-beta was expressed in the dermis. Inhibitors of the TGF-beta signal pathway partially inhibited transdifferentiation. Overexpression of both hTG2/Gh and mGbx1 together in the epidermis by electroporation resulted in cuboidal cells in the upper cell layers of the epidermis without keratinized layers, although epidermal keratinization was observed in skin by overexpression of either of them. Labeling DNA with BrdU indicated that RA directly transdifferentiated transient amplifying epidermal cells, not stem cells, to mucosal cells. This study showed that coexpression of TG/2 and Gbx1 in the epidermis was required for esophagus-like mucosal transdifferentiation, and that increase in TGF-beta2 expression by RA in the dermis was essential to induce transdifferentiation through epithelial-mesenchymal interaction.     

Reactive oxygen species promotes cellular senescence in normal human epidermal keratinocytes through epigenetic regulation of p16

Reactive oxygen species (ROS) can cause severe damage to DNA, proteins and lipids in normal cells, contributing to carcinogenesis and various pathological conditions. While cellular senescence arrests the early phase of cell cycle without any detectable telomere loss or dysfunction. ROS is reported to contribute to induction of cellular senescence, as evidence by its premature onset upon treatment with antioxidants or inhibitors of cellular oxidant scavengers. Although cellular senescence is known to be implicated in tumor suppression, it remains unknown whether ROS initially contributed to be cellular senescence in normal human epidermal keratinocytes (NHEK) and their malignant counterparts. To clarify whether ROS induce cellular senescence in NHEKs, we examined the effect of hydrogen peroxide (H₂O₂) on the expression of cellular senescence-associated molecules in NHEKs, compared to in squamous carcinoma cells (SCCs). Hydrogen peroxide increased the number of cells positive in senescence associated-β-galactosidase (SA-β-Gal) activity in NHEKs, but not SCCs. The expression of cyclin-dependent kinase (CDK) inhibitors, especially p16^INK4a was upregulated in NHEKs treated with H₂O₂. Interestingly, H₂O₂ suppressed the methylation of p16^INK4a, promoter region in NHEKs, but not in SCCs. Hydrogen peroxide also suppressed the expression of phosphorylated Rb and CDK4, resulting in arrest in G0/G1 phase in NHEKs, but not SCCs.     

A model for the hierarchical structure of the human epidermal cornified cell envelope

The epidermal cornified cell envelope (CE) is a 15 nm thick layer of highly insoluble protein that is assembled on the intracellular surface of the cell membrane during terminal differentiation, and comprises about 10% of the mass of the cornified dead layers of the tissue. The CE consists of a complex amalgam of several known proteins that are crosslinked by isodipeptide bonds formed by the action of transglutaminases, but little is known about their order of accretion during CE assembly, or how they are crosslinked. In this paper, CEs purified from human foreskin epidermis were examined by immunogold electron microscopy before and after digestion with proteases.The mass fractions of the proteins remaining in CE remnants during digestion were estimated from the amino acid compositions by mathematical modelling. Together, the data support a new model for the complex hierachical structure of the CE. The cytoplasmic surface of intact purified CEs consists of filaggrin, loricrin, SPRs and keratin intermediate filaments. The bulk of the CE consists of a mixture of loricrin (75%) and SPRs (5%). Following removal of most of these, the novel protein elafin is exposed, which contributes about 6% of CE mass. The protein material on the inner CE 'core' adjacent or attached to the lipid envelope consists of cystatin alpha (5%), involucrin (2%), keratin filaments (3%) and possibly other as yet unidentified protein(s)(2-5%). This model supports but considerably extends an earlier extant hypotheis for CE structure, and thus provides the basis for further detailed biochemical and ultra-structural studies.     

Initiation of assembly of the cell envelope barrier structure of stratified squamous epithelia

The cell envelope (CE) is a specialized structure that is important for barrier function in terminally differentiated stratified squamous epithelia. The CE is formed inside the plasma membrane and becomes insoluble as a result of cross-linking of constituent proteins by isopeptide bonds formed by transglutaminases. To investigate the earliest stages of assembly of the CE, we have studied human epidermal keratinocytes induced to terminally differentiate in submerged liquid culture as a model system for epithelia in general. CEs were harvested from 2-, 3-, 5-, or 7-d cultured cells and examined by 1) immunogold electron microscopy using antibodies to known CE or other junctional proteins and 2) amino acid sequencing of cross-linked peptides derived by proteolysis of CEs. Our data document that CE assembly is initiated along the plasma membrane between desmosomes by head-to-tail and head-to-head cross-linking of involucrin to itself and to envoplakin and perhaps periplakin. Essentially only one lysine and two glutamine residues of involucrin and two glutamines of envoplakin were used initially. In CEs of 3-d cultured cells, involucrin, envoplakin, and small proline-rich proteins were physically located at desmosomes and had become cross-linked to desmoplakin, and in 5-d CEs, these three proteins had formed a continuous layer extending uniformly along the cell periphery. By this time >15 residues of involucrin were used for cross-linking. The CEs of 7-d cells contain significant amounts of the protein loricrin, typically expressed at a later stage of CE assembly. Together, these data stress the importance of juxtaposition of membranes, transglutaminases, and involucrin and envoplakin in the initiation of CE assembly of stratified squamous epithelia.     

Upregulation of P2Y2 receptors by retinoids in normal human epidermal keratinocytes

Retinoids, vitamin A derivatives, are important regulators of the growth and differentiation of skin cells. Although retinoids are therapeutically used for several skin ailments, little is known about their effects on P2 receptors, known to be involved in various functions in the skin. DNA array analysis showed that treatment of normal human epidermal keratinocytes (NHEKs) with all-trans-retinoic acid (ATRA), an agonist to RAR (retinoic acid receptor), enhanced the expression of mRNA for the P2Y2 receptor, a metabotropic P2 receptor that is known to be involved in the proliferation of the epidermis. The expression of other P2 receptors in NHEKs was not affected by ATRA. ATRA increased the mRNA for the P2Y2 receptor in a concentration-dependent fashion (1 nM to 1 μM). Am80, a synthesized agonist to RAR, showed a similar enhancement, whereas 9-cis-retinoic acid (9-cisRA), an agonist to RXR (retinoid X receptor), enhanced P2Y2 gene expression to a lesser extent. Ca²⁺ imaging analysis showed that ATRA also increased the function of P2Y2 receptors in NHEKs. Retinoids are known to enhance the turnover of the epidermis by increasing both proliferation and terminal differentiation. The DNA microarray analysis also revealed that ATRA upregulates various genes involved in the differentiation of NHEKs. Our present results suggest that retinoids, at least in part, exert their proliferative effects by upregulating P2Y2 receptors in NHEKs. This effect of retinoids may be closely related to their therapeutic effect against various ailments or aging events in skins such as over-keratinization, pigmentation and re-modeling.     

Teratocarcinoma F9 cells induced to differentiate with sodium butyrate produce both tissue-type and urokinase-type plasminogen activators.

Sodium butyrate (NaB) can induce teratocarcinoma cell differentiation as retinoic acid (RA). However, the function of these two agents seems to be a little different [Kosaka et al., Exp Cell Res, 192:46-51, 1991]. F9 cells treated with NaB synthesize both tissue-type (tPA) and urokinase-type (uPA) plasminogen activator, though RA induces only tPA production. Urokinase-type PA is demonstrated to exist in association with membrane and to localize its activity to the close environment of the cell surface. This may cause the specific cell morphology and characteristics of differentiated F9 cells induced with NaB.     

Impaired ability of burn patient neutrophils to stimulate β-defensin production by keratinocytes

Immunosuppressive neutrophils (PMN-II) appearing in association with burn injury have a role on the increased susceptibility of burn patients to various infections. In the present study, the role of PMN-II on the production of human β-defensins (HBDs), important molecules on host antimicrobial innate immunities, by human keratinocytes was studied. Normal human epidermal keratinocytes (NHEKs) were cultured with neutrophils (PMNs) isolated from burn patients or healthy volunteers in dual-chamber transwells. Culture fluids harvested 24?h after cultivation were assayed for HBDs using enzyme-linked immunosorbent assay. Also, these culture fluids were assayed for their antimicrobial activities by a standard colony-counting method using Pseudomonas aeruginosa. In the results, PMNs isolated from peripheral blood of burn patients were confirmed as PMN-II, because these cells produced CC-chemokine ligand 2 (CCL2), but not interleukin (IL)-12 and CC-chemokine ligand 3 (CCL3). Culture fluids of NHEK transwell-cultured with healthy PMNs exhibited strong killing activities against P. aeruginosa (96% inhibition), however, the growth of bacteria was not dramatically inhibited by the culture fluids of NHEK transwell-cultured with burn-patient PMNs (36% inhibition). IL-12 and CCL3 containing culture fluids of healthy PMNs stimulated with the bacterial antigen or rCCL3 and rIL-12 enhanced the production of HBD2 and HBD3 by NHEK, whereas CCL2 containing culture fluids of burn-patient PMN stimulated with the antigen or rCCL2 inhibited the HBD production by NHEK. These results indicate that PMN-II appearing in association with burn injury contribute to the decreased production of HBDs in thermally injured patients.     

Lipin-1 expression is critical for keratinocyte differentiation

Lipin-1 is an Mg²⁺-dependent phosphatidate phosphatase that facilitates the dephosphorylation of phosphatidic acid to generate diacylglycerol. Little is known about the expression and function of lipin-1 in normal human epidermal keratinocytes (NHEKs). Here, we demonstrate that lipin-1 is present in basal and spinous layers of the normal human epidermis, and lipin-1 expression is gradually downregulated during NHEK differentiation. Interestingly, lipin-1 knockdown (KD) inhibited keratinocyte differentiation and caused G1 arrest by upregulating p21 expression. Cell cycle arrest by p21 is required for commitment of keratinocytes to differentiation, but must be downregulated for the progress of keratinocyte differentiation. Therefore, reduced keratinocyte differentiation results from sustained upregulation of p21 by lipin-1 KD. Lipin-1 KD also decreased the phosphorylation/activation of protein kinase C (PKC)α, whereas lipin-1 overexpression increased PKCα phosphorylation. Treatment with PKCα inhibitors, like lipin-1 KD, stimulated p21 expression, while lipin-1 overexpression reduced p21 expression, implicating PKCα in lipin-1-induced regulation of p21 expression. Taken together, these results suggest that lipin-1-mediated downregulation of p21 is critical for the progress of keratinocyte differentiation after the initial commitment of keratinocytes to differentiation induced by p21, and that PKCα is involved in p21 expression regulation by lipin-1.