Changes in keratin gene expression during terminal differentiation of the keratinocyte

Cells of the inner layers of the epidermis contain small keratins (46-58K), whereas the cells of the outer layers contain large keratins (63-67K) in addition to small ones. The changes in keratin composition that take place within each cell during the course of its terminal differentiation result largely from changes in synthesis. Cultured epidermal cells resemble cells of the inner layers of the epidermis in synthesizing only small keratins. The cultured cells possess translatable mRNA only for small keratins, whereas mRNA extracted from whole epidermis can be translated into both large and small keratins. As no synthesis takes place in the outermost layer of the epidermis (stratum corneum), the keratins of this layer must be synthesized earlier, but in some cases they then become smaller: this presumably occurs by post-translational processing of the molecules during the final stages of differentiation. Stratified squamous epithelia of internal organs do not form a typical stratum corneum and do not make the large keratins characteristic of epidermis. Their keratins are also different from those of cultured keratinocytes, implying that they have embarked on an alternate route of terminal keratin synthesis.     

Ceramide-dependent regulation of human epidermal keratinocyte CD1d expression during terminal differentiation

Human keratinocytes (KC), when cultured under conditions to remain undifferentiated or to terminally differentiate, changed their cellular distribution of CD1d. As studied by confocal microscopy, undifferentiated KC had a pool of cytoplasmic CD1d, whereas after terminal differentiation, this molecule localized in the cell membrane, which recapitulates CD1d expression in vivo. A comparison of undifferentiated and differentiated cultured KC did not reveal any differences in the association with beta(2)-microglobulin, invariant chain of class II MHC, or patterns of glycosylation, suggesting that these biochemical properties are not regulating the cellular distribution of CD1d. Time-course studies of CD1d gene expression indicated that KC slowly increased gene expression with CaCl(2)-induced terminal differentiation. Increased CD1d gene expression was dependent on ceramide synthesis, because fumonisin B1, a ceramide synthetase inhibitor, blocked the increase in CD1d gene expression during terminal differentiation. Similarly, exogenous ceramide or the ceramidase inhibitor, B13, induced CD1d gene expression by undifferentiated, but not terminally differentiated, KC. A protein kinase C-zeta (PKC-zeta) inhibitor (a pseudosubstrate oligopeptide), but not a PKC-alphabeta inhibitor, significantly decreased CD1d gene expression by undifferentiated or ceramide-stimulated cultured, undifferentiated KC. As expected, downstream signaling events of PKC-zeta (JNK phosphorylation and NF-kappaBeta accumulation in the nucleus) were also attenuated. The calcineurin phosphatase inhibitor cyclosporine A, which blocks KC terminal differentiation, also blocked CD1d gene expression by cultured KC. In conclusion, this novel function of cellular ceramides extends the importance of this class of biologically active lipids beyond that of terminal differentiation and barrier function in normal human skin.     

Changes in integrin expression during adipocyte differentiation

3T3-L1 preadipocytes require cAMP for maximal differentiation. Microarray analysis reveals that the integrins alpha5 and alpha6 are coordinately regulated by cAMP. alpha5 expression is gradually diminished during adipogenesis, whereas alpha6 is increased. Overexpression of alpha5 in preadipocytes results in enhanced proliferation and attenuated differentiation. Conversely, alpha6 overexpression is without effect. The GTPase Rac is normally inhibited during differentiation. However, overexpression of integrin alpha5 increases Rac activity. Constitutively active but not dominant-negative Rac inhibits differentiation when overexpressed in preadipocytes, implying its role downstream of alpha5 integrin in maintaining preadipocytes in an undifferentiated state. Moreover, alpha6 integrin is critically involved in clustering growth-arrested preadipocytes on basement membrane Matrigel. Perturbation of such clustering enhances Rho activity and promotes growth-arrested preadipocytes to reenter the cell cycle. These findings demonstrate a role for integrin alpha6 in connecting morphogenesis with signaling processes leading to terminal differentiation.     

Multiple pathways are involved in DNA degradation during keratinocyte terminal differentiation

Loss of the nucleus is a critical step in keratinocyte terminal differentiation. To elucidate the mechanisms involved, we focused on two characteristic events: nuclear translocation of N-terminal fragment of profilaggrin and caspase-14-dependent degradation of the inhibitor of caspase-activated DNase (ICAD). First, we demonstrated that epidermal mesotrypsin liberated a 55-kDa N-terminal fragment of profilaggrin (FLG-N) and FLG-N was translocated into the nucleus. Interestingly, these cells became TUNEL positive. Mutation in the mesotrypsin-susceptible Arg-rich region between FLG-N and the first filaggrin domain abolished these changes. Furthermore, caspase-14 caused limited proteolysis of ICAD, followed by accumulation of caspase-activated DNase (CAD) in TUNEL-positive nuclei. Knockdown of both proteases resulted in a significant increase of remnant nuclei in a skin equivalent model. Immunohistochemical study revealed that both caspase-14 and mesotrypsin were markedly downregulated in parakeratotic areas of lesional skin from patients with atopic dermatitis and psoriasis. Collectively, our results indicate that at least two pathways are involved in the DNA degradation process during keratinocyte terminal differentiation.     

Evidence that terminal deoxynucleotidyltransferase expression plays a role in Ig heavy chain gene segment utilization

TdT is a nuclear enzyme that catalyzes the addition of random nucleotides at Ig and TCR V(D)J junctions. In this paper we analyze human IgH rearrangements generated from transgenic minilocus mice in the presence or absence of TdT. In the absence of TdT, the pseudo-VH gene segment present in the minilocus is rearranged dramatically more frequently. Additionally, JH6 gene segment utilization is increased as well as the number of rearrangements involving only VH and JH gene segments. Thus, the recombination of IgH gene segments that are flanked by 23-nt spacer recombination signal sequences may be influenced by TdT expression. Extensive analysis indicates that these changes are independent of antigenic selection and cannot be explained by homology-mediated recombination. Thus, the role played by TdT may be more extensive than previously thought.     

Changes in keratinocyte adhesion during terminal differentiation: reduction in fibronectin binding precedes alpha 5 beta 1 integrin loss from the cell surface

During terminal differentiation keratinocytes move out of the basal layer of the epidermis and thereby lose contact with the basement membrane. We show that terminal differentiation in culture involves loss of adhesiveness to fibronectin, laminin, and collagen types I and IV. The adhesive changes precede, by several hours, loss of the alpha 2 beta 1, alpha 3 beta 1, and alpha 5 beta 1 integrins from the cell surface. Keratinocyte adhesion to fibronectin is mediated by the alpha 5 beta 1 integrin, and the decrease in adhesion of intact cells to fibronectin is correlated with a decrease in the ability of alpha 5 beta 1 receptors to bind fibronectin. Thus modulation of integrin function early in terminal differentiation may be an early event determining cell migration out of the basal layer.     

Evidence that specific interactions play a role in the cholesterol sensitivity of G protein-coupled receptors

G protein-coupled receptors (GPCRs) are known to be modulated by membrane cholesterol levels, but whether or not the effects are caused by specific receptor-cholesterol interactions or cholesterol's general effects on the membrane is not well-understood. We performed coarse-grained molecular dynamics (CGMD) simulations coupled with structural bioinformatics approaches on the β₂-adrenergic receptor (β₂AR) and the cholecystokinin (CCK) receptor subfamily. The β₂AR has been shown to be sensitive to membrane cholesterol and cholesterol molecules have been clearly resolved in numerous β₂AR crystal structures. The two CCK receptors are highly homologous and preserve similar cholesterol recognition motifs but despite their homology, CCK₁R shows functional sensitivity to membrane cholesterol while CCK₂R does not. Our results offer new insights into how cholesterol modulates GPCR function by showing cholesterol interactions with β₂AR that agree with previously published data; additionally, we observe differential and specific cholesterol binding in the CCK receptor subfamily while revealing a previously unreported Cholesterol Recognition Amino-acid Consensus (CRAC) sequence that is also conserved across 38% of class A GPCRs. A thermal denaturation assay (LCP-T_m) shows that mutation of a conserved CRAC sequence on TM7 of the β₂AR affects cholesterol stabilization of the receptor in a lipid bilayer. The results of this study provide a better understanding of receptor-cholesterol interactions that can contribute to novel and improved therapeutics for a variety of diseases.     

Glucose metabolism in cancer. Evidence that demethylation events play a role in activating type II hexokinase gene expression

One of the "signature" phenotypes of highly malignant, poorly differentiated tumors, including hepatomas, is their remarkable propensity to utilize glucose at a much higher rate than normal cells, a property frequently dependent on the marked overexpression of type II hexokinase (HKII). As the expression of the gene for this enzyme is nearly silent in liver tissue, we tested the possibility that DNA methylation/demethylation events may be involved in its regulation. Initial studies employing methylation restriction endonuclease analysis provided evidence for differential methylation patterns for the HKII gene in normal hepatocytes and hepatoma cells, the latter represented by a highly glycolytic model cell line (AS-30D). Subsequently, sequencing following sodium bisulfite treatment revealed 18 methylated CpG sites within a CpG island (-350 to +781 bp) in the hepatocyte gene but none in that of the hepatoma. In addition, treatment of a hepatocyte cell line with the DNA methyltransferase inhibitors, 5'-azacytidine and 5'-aza-2'-deoxycytidine, activated basal expression levels of HKII mRNA and protein. Finally, stably transfecting the hepatocyte cell line with DNA demethylase also resulted in activating the basal expression levels of HKII mRNA and protein. These novel observations indicate that one of the initial events in activating the HKII gene during either transformation or tumor progression may reside at the epigenetic level.     

Evidence that microtubules play a permissive role in hepatocyte very low density lipoprotein secretion

To determine whether a minimum number of assembled microtubules is required for very low density lipoprotein (VLDL) triglyceride TG) secretion in hepatocytes, antimicrotubule drugs of different concentrations were given to rats. Hepatic VLDL-TG release was subsequently measured by a liver perfusion system, and hepatocyte ultrastructural changes were analyzed by quantitative ultrastructural methods. The results demonstrate a tight coupling between the reduction in hepatocyte microtubule content and the reduction in hepatic VLDL-TG secretion which is related to the dose of colchicine or vinblastine administered. The various estimates imply that a minimum number of microtubules is necessary for hepatic VLDL secretion to proceed normally and that hepatic VLDL secretion rates reach their nadir (10-- 30% of control) when microtubules comprise less than 0.005% of the cytoplasm (or less than 10% of control values) when microtubules comprise less than 0.005% of the cytoplasm (or less than 10% of control values). At this point, hepatocyte Golgi complexes are also greatly altered; Golgi complexes with recognizable dictyosomal membranes are reduced to 15% of control values and the region is filled with large numbers of electron-dense bodies which appear to be lysosomes in the process of digesting VLDL. There is a predilection for the remaining Golgi complexes to be associated with a few segments of microtubules, even when no microtubules can be measured in random samplings of hepatocytes. Clusters of vacuoles containing VLDL are also present throughout the cytoplasm; the limiting membranes of 25% of these vacuoles are studded with ribosomes. These findings demonstrate that the administration of antimicrotubule agents results in decreases in hepatic VLDL-TG secretion which are associated with loss of microtubules and alteration of existing Golgi complexes.     

Plasminogen activator inhibitor 2: expression and role in differentiation of epidermal keratinocyte

Plasminogen activator inhibitor 2 (PAI-2) is an enzyme inhibitor which is involved in cell differentiation, tissue growth and regeneration. In this study, immunocytochemistry, in situ hybridization and confocal laser scanning microscopy were used to investigate the expression and role of PAI-2 in differentiation of keratinocytes in vitro. The result showed that in the mono-layer differentiated keratinocytes induced by high calcium concentration, the expression of PAI-2 and its mRNA increased significantly, accompanied by expression increase of the differentiation marker keratin 10; and in the multi-layer differentiated keratinocytes induced by high calcium, PAI-2 expressed strongly mainly in the keratinocytes of middle as well as upper stratified layers, while K10 expressed in the keratinocytes of all stratified layers. Furthermore, the changes of the parameters related to keratinocyte differentiation were detected after inhibition of PAI-2 functions by its antibody, and the data showed that when treated by PAI-2 antibody, involucrin in the keratinocytes envelope expressed increasingly with an altering distribution from part to the whole envelope area. Our results indicate that during differentiation of epidermal keratinocyte, PAI-2 expresses mainly in the more differentiated keratinocytes and may protect the terminal differentiated keratinocytes from prematuration through inhibiting involucrin expression in cornified envelope.     

Evidence that phospholipids play a key role in pre-beta apoA-I formation and high-density lipoprotein remodeling

The initial plasma acceptor of unesterified cholesterol and phospholipids from peripheral cells has been identified as pre-beta migrating, lipid-free, or lipid-poor apolipoprotein (apo) A-I (pre-beta apoA-I). Pre-beta apoA-I is formed when plasma factors, such as cholesteryl ester transfer protein (CETP), remodel high-density lipoproteins (HDL). The aim of this study is to determine how phospholipids influence pre-beta apoA-I formation during the CETP-mediated remodeling of HDL. Reconstituted HDL (rHDL) containing either 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC), 1-palmitoyl-2-linoleoyl phosphatidylcholine (PLPC), 1-palmitoyl-2-arachidonyl phosphatidylcholine (PAPC), or 1-palmitoyl-2-docosahexanoyl phosphatidylcholine (PDPC) as the only phospholipid were prepared. The rHDL were comparable in size and core lipid/protein molar ratio and contained only cholesteryl esters in their core and apoA-I as the sole apolipoprotein. The (POPC)rHDL, (PLPC)rHDL, (PAPC)rHDL, and (PDPC)rHDL were respectively incubated for 0-24 h with CETP and microemulsions containing triolein and either POPC, PLPC, PAPC, or PDPC. The rate at which the rHDL were depleted of core lipids and remodeled to small particles varied widely with (POPC)rHDL < (PLPC)rHDL < (PDPC)rHDL approximately (PAPC)rHDL. Pre-beta apoA-I was not formed in the (POPC)rHDL incubations. Pre-beta apoA-I was apparent by 24 h in the (PLPC)rHDL incubations and by 12 h in the (PAPC)rHDL and (PDPC)rHDL incubations. The enhanced formation of pre-beta apoA-I in the (PAPC)rHDL and (PDPC)rHDL incubations reflected the increased core lipid depletion of the particles combined with the destabilization and progressive exclusion of apoA-I from the particle surface. In conclusion, these results show that phospholipids play a key role in the CETP-mediated remodeling of rHDL and pre-beta apoA-I formation.     

Evidence that receptor aggregation may play a role in transmembrane signaling through the insulin-like growth factor-I receptor

alpha IR-3 is a mouse monoclonal antibody that binds to an epitope on the human insulin-like growth factor I (IGF-I) receptor and inhibits [125I]IGF-I binding to this receptor on human skin fibroblasts (HSF) and Hep G2 human hepatoblastoma cells. Unlike the natural ligand (IGF-I), neither intact alpha IR-3 nor its monovalent Fab fragment stimulate aminoisobutyric acid (AIB) uptake in HSF, and both competitively antagonize IGF-I's ability to produce this effect. However, when HSF are incubated with alpha IR-3 or its Fab' fragment, subsequent exposure to anti-mouse immunoglobulin G (IgG) produces a potent stimulation of AIB uptake. Anti-Mouse IgG by itself does not effect AIB uptake. alpha IR-3 also antagonizes IGF-I's ability to stimulate glycogen synthesis in Hep G2 cells. As with AIB uptake in HSF, the combination of alpha IR-3 followed by anti-mouse IgG stimulates glycogen synthesis in Hep G2 cells to the same extent as that produced by IGF-I. The triggering of these two biological effects depends on the concentration of both alpha IR-3 and anti-mouse IgG. These results are consistent with the possibility that local aggregation or cross-linking of IGF-I receptors plays an important role in transmembrane signaling by this receptor.     

Epithelial-specific gene expression during differentiation of stratified primary human keratinocyte cultures.

Cultured epithelial cells are used to generate extensive patches of autologous skin equivalent for patients with burns or wounds and to investigate the growth and differentiation of epithelia in vitro. We have undertaken a comprehensive study of the morphological and molecular events that occur during culturing of human foreskin keratinocytes at the liquid-air interface on a dermal equivalent consisting of a collagen matrix containing fibroblasts. Using radioactively labeled RNA probes for mRNAs and monoclonal antibodies for proteins, we found that the expression of a comprehensive set of differentiation stage-specific genes was affected by the type of fibroblasts included in the matrix as well as by the age of the culture. The expression of these genes was not always coordinated and could not be predicted from the histological appearance of the stratified epithelium. Surprisingly, the mouse fibroblasts promoted epithelial differentiation much more closely resembling foreskin than did the homologous primary foreskin fibroblasts.     

Variable expression of some "housekeeping" genes during human keratinocyte differentiation

We investigated the expression levels of four cellular "housekeeping" genes during epithelial differentiation. Differentiation is a dynamic process and various cellular RNAs have been targeted for use as internal controls during differentiation of human keratinocytes, but the consistent expression of such standards has not been previously validated. We used the organotypic (raft) culture system to grow stratified and differentiated epithelium in vitro. We compared cellular RNAs from epithelial tissues of both normal human keratinocytes and keratinocytes whose differentiation scheme is altered by the replication of human papillomavirus. Using ribonuclease protection assays to quantify RNA expression levels, we found that beta-actin and glyceraldehyde-3-phosphate dehydrogenase levels fluctuated during epithelial differentiation, whereas cyclophilin RNA and 28S-ribosomal RNA were the most consistently expressed during epithelial differentiation. These stably expressed cellular RNAs can be targeted as controls to permit quantitative expression analyses of cellular and pathogen RNAs during epithelial differentiation under various experimental conditions.