Publications

At birth, neonatal stratum corneum (SC) pH is close to neutral but acidifies with maturation, which can be ascribed, in part, to secretory phospholipase A(2) and sodium/hydrogen antiporter 1 (NHE1) activities. Here we assessed the functional consequences of a neutral SC pH in a newborn rat model. While basal transepidermal water loss rates are near normal, barrier recovery (BR) rates after acute barrier disruption were delayed in newborn animals. The abnormality in barrier homeostasis could be improved by topical applications of an acidic buffer, indicating that barrier abnormality is primarily due to high SC pH. The delay in BR correlated with incompletely processed lamellar membranes and decreased activity of beta-glucocerebrosidase. Inhibition of NHE1 delayed BR after acute barrier perturbation. SC integrity was abnormal in newborn animals. Electron microscopy demonstrated decreased corneodesmosomes (CD) in newborn animals with decreased expression of desmoglein 1 and corneodesmosin. Serine protease activation appears to be responsible for CD degradation in newborn animals, because serine protease activity is increased in the SC and it can be reduced by acidification of the SC. The delay in acidification of neonatal SC results in abnormalities in permeability barrier homeostasis and SC integrity and are likely due to pH-induced modulations in enzyme activity.

Previous studies demonstrated that peroxisome-proliferator-activated receptor (PPAR)-alpha or PPAR-delta activation stimulates keratinocyte differentiation, is anti-inflammatory, and improves barrier homeostasis. Here we demonstrate that treatment of cultured human keratinocytes with ciglitazone, a PPAR-gamma activator, increases involucrin and transglutaminase 1 mRNA levels. Moreover, topical treatment of hairless mice with ciglitazone or troglitazone increases loricrin, involucrin, and filaggrin expression without altering epidermal morphology. These results indicate that PPAR-gamma activation stimulates keratinocyte differentiation. Additionally, PPAR-gamma activators accelerated barrier recovery following acute disruption by either tape stripping or acetone treatment, indicating an improvement in permeability barrier homeostasis. Treatment with PPAR-gamma activators also reduced the cutaneous inflammatory response that is induced by phorbol 12-myristate-13-acetate, a model of irritant contact dermatitis and oxazolone, a model of allergic contact dermatitis. To determine whether the effects of PPAR-gamma activators are mediated by PPAR-gamma, we next examined animals deficient in PPAR-gamma. Mice with a deficiency of PPAR-gamma specifically localized to the epidermis did not display any cutaneous abnormalites on inspection, but on light microscopy there was a modest increase in epidermal thickness associated with an increase in proliferating cell nuclear antigen (PCNA) staining. Key functions of the skin including permeability barrier homeostasis, stratum corneum surface pH, and water-holding capacity, and response to inflammatory stimuli were not altered in PPAR-gamma-deficient epidermis. Although PPAR-gamma activators stimulated loricrin and filaggrin expression in wild-type animals, however, in PPAR-gamma-deficient mice no effect was observed indicating that the stimulation of differentiation by PPAR-gamma activators is mediated by PPAR-gamma. In contrast, PPAR-gamma activators inhibited inflammation in both PPAR-gamma-deficient and wild-type mouse skin, indicating that the inhibition of cutaneous inflammation by these PPAR-gamma activators does not require PPAR-gamma in keratinocytes. These observations suggest that thiazolidindiones and perhaps other PPAR-gamma activators maybe useful in the treatment of cutaneous disorders.

UNLABELLED

Type 2 diabetes may be associated with elevated fracture risk, but the impact on bone loss is unknown. Analysis of 4-year change in hip BMD data from a cohort of white and black well-functioning men and women 70-79 years of age found that white women with diabetes had more rapid bone loss at the femoral neck than those with normal glucose metabolism.

INTRODUCTION

Type 2 diabetes may be associated with elevated fracture risk in older adults. Although type 2 diabetes is not associated with lower BMD, older diabetic adults have a higher prevalence of other risk factors for fracture, including more frequent falls, functional limitations, and diabetic complications. With this burden of risk factors, loss of BMD could place older adults with diabetes at higher risk of sustaining a fracture.

MATERIALS AND METHODS

To determine if bone loss is increased with type 2 diabetes, we analyzed data from the Health, Aging, and Body Composition Study of white and black well-functioning men and women 70-79 years of age. Hip BMD was measured at baseline and 4 years later in 480 (23%) participants with diabetes, 439 with impaired glucose metabolism, and 1172 with normal glucose homeostasis (NG).

RESULTS

Those with diabetes had higher baseline hip BMD and weight, but among white women, had more weight loss over 4 years. White women with diabetes lost more femoral neck and total hip BMD than those with NG in age-adjusted models. After multivariable adjustment, diabetes was associated with greater loss of femoral neck BMD (-0.32%/year; 95% CI: -0.61, -0.02) but not total hip BMD. In men and black women, change in hip BMD was similar for participants with diabetes and NG.

CONCLUSIONS

Despite having higher baseline BMD, diabetic white women, but not men or black women, had more rapid bone loss at the femoral neck than those with NG. This increased bone loss may contribute to the higher fracture risk observed in older diabetic women.

Although many skin disorders, including psoriasis and atopic dermatitis, are adversely affected by psychologic stress (PS), the pathophysiologic link between PS and disease expression remains unclear. Recent studies demonstrated PS-induced alterations in permeability barrier homeostasis, mediated by increased endogenous glucocorticoids. Here, we assessed the mechanisms by which PS alters stratum corneum (SC) function. Insomniac psychologic stress (IPS) altered both barrier homeostasis and SC integrity. IPS decreased epidermal cell proliferation, impaired epidermal differentiation, and decreased the density and size of corneodesmosomes (CD), which was linked to degradation of CD proteins (e.g., desmoglein1). Barrier compromise was linked to decreased production and secretion of lamellar bodies (LB), which in turn could be attributed to a decrease in de novo synthesis of epidermal lipids. Topical physiologic lipids (equimolar cholesterol, ceramides, and free fatty acids) normalized both barrier homeostasis and SC integrity in IPS mice, further evidence that lipid deficiency accounted for these functional abnormalities. Thus, PS inhibition of epidermal lipid synthesis results in decreased LB formation and secretion, as well as decreased CD, compromising both permeability barrier homeostasis and SC integrity. These studies suggest that topical treatment with epidermal physiologic lipids could be beneficial in stress-induced, barrier-associated dermatoses, such as psoriasis and atopic dermatitis.

Although stratum corneum (SC) hydration has been primarily of concern to the cosmetic industry, it serves an important biosensor function. In murine models, not only deiminated products of filaggrin-derived amino acids ("NMF") but also endogenous glycerol from circulation into the epidermis via aquaporin 3 channel and from triglyceride turnover in sebaceous glands (SG) are important determinants. We assessed here whether endogenous glycerol could also be linked to SC hydration in humans. SG-enriched sites are more hydrated than SG-impoverished sites, and SC hydration correlates with both sebum production and SC glycerol content, but the correlation is more significant for SC glycerol content than for sebum content. Moreover, gender-related differences in sebum content are not associated with altered SC hydration. SC hydration is also linked to SC glycerol content in SG-impoverished sites, suggesting a role for non-SG-derived (? from circulation) glycerol in SC hydration. Finally, short-term water immersion produces a parallel decline in SC hydration and SC glycerol content, with glycerol levels returning to normal over several hours. These results suggest that endogenous glycerol of both circulatory and SG origin comprises an H2O-extractable pool that influences SC hydration in humans. These results also provide a rationale for the development of glycerol-containing therapeutic moisturizers.

We showed recently that short-term increases in stratum corneum (SC) pH are accompanied by minor alterations in permeability barrier homeostasis and SC integrity/cohesion. Since prolonged SC neutralization more closely mirrors clinical situations (i.e., neonatal skin, occupational dermatitis conditions), we assessed here whether sustained elevations of SC pH by long-term application of 1,1,3,3-tetramethylguanidine superbase provoke profound alterations in SC function. Sustained SC neutralization altered not only barrier recovery kinetics but also basal permeability barrier function. These abnormalities were attributable to a decrease in beta-glucocerebrosidase (beta-GlcCer'ase) and acidic sphingomyelinase (aSMase) catalytic activity and enzyme degradation consequent to a pH-induced sustained serine protease (SP) activity. The role of SP in this process was shown by the normalization of enzyme activities/content by co-applied SP inhibitors (SPI). To address whether lipid-processing enzymes are potential substrates for the stratum corneum chymotryptic enzyme (SCCE), protein extracts from human SC were treated for 2 h at 37 degrees C with recombinant active SCCE at pH 7.2. Recombinant SCCE induced a significant decrease in the immunoblotting of both beta-GlcCer'ase or aSMase compared with control experiments performed in the absence of the active SCCE. Finally, with sustained SC neutralization, SC integrity/cohesion deteriorated, attributable to SP-mediated degradation of corneodesmosomes (CD) as well as CD constituent proteins, desmoglein 1. These abnormalities were again reversed by co-applied SPI. In conclusion, prolonged SC neutralization provokes profound abnormalities in SC function, due to pH-induced high SP activity that, in turn, degrades lipid processing enzymes and CD proteins.

Liver X receptors (LXRs) and peroxisome proliferator-activated receptors (PPARs) are potent regulators of keratinocyte proliferation, differentiation, and epidermal permeability barrier homeostasis. Cholesterol sulfotransferase type 2B isoform 1b (SULT2B1b) is a key enzyme in the synthesis of cholesterol sulfate (CS), a critical regulator of keratinocyte differentiation and desquamation, as well as a mediator of barrier homeostasis. In this study, we assessed the effect of activators of LXR, PPARalpha, PPARbeta/delta, and PPARgamma on SULT2B1b gene expression and enzyme activity in cultured human keratinocytes (CHKs). Our results demonstrate that PPAR and LXR activators increase SULT2B1b mRNA levels, with the most dramatic effect (a 26-fold increase) induced by the PPARgamma activator ciglitazone. Ciglitazone upregulates SULT2B1b mRNA in a dose- and time-dependent manner. Moreover, the stimulation of SULT2B1b gene expression by LXR and PPAR activators occurs in both undifferentiated and differentiated CHKs. The upregulation of SULT2B1b mRNA by ciglitazone appears to occur at a transcriptional level, because the degradation of SULT2B1b is not accelerated by ciglitazone. In addition, cycloheximide almost completely blocks the ciglitazone-induced increase in SULT2B1b mRNA, suggesting that the transcription of SULTB1b mRNA is dependent on new protein synthesis. Finally, LXR and PPAR activators also increased the activity of cholesterol sulfotransferase. Thus, LXR and PPAR activators regulate the expression of SULT2B1b, the key enzyme in the synthesis of CS, which is a potent regulator of epidermal differentiation and corneocyte desquamation.

Phospholipids are a major class of lipids in epidermis, where they serve as a source of free fatty acids that are important for the maintenance of epidermal permeability barrier function. The phospholipid biosynthetic enzyme, 1-acyl-sn-glycerol-3-phosphate acyltransferase (AGPAT), catalyzes the acylation of lysophosphatidic acid to form phosphatidic acid, the major precursor of all glycerolipids. We identified an expression pattern of AGPAT isoforms that is unique to epidermis, with relatively high constitutive expression of mouse AGPAT (mAGPAT) 3, 4, and 5 but low constitutive expression of mAGPAT 1 and 2. Localization studies indicate that all five isoforms of AGPAT were expressed in all nucleated layers of epidermis. Furthermore, rat AGPAT 2 and 5 mRNAs increased in parallel with both an increase in enzyme activity and permeability barrier formation late in rat epidermal development. Moreover, after two methods of acute permeability barrier disruption, mAGPAT 1, 2, and 3 mRNA levels increased rapidly and were sustained for at least 24 h. In parallel with the increase in mRNA levels, an increase in AGPAT activity also occurred. Because upregulation of mAGPAT mRNAs after tape-stripping could be partially reversed by artificial barrier restoration by occlusion, these studies suggest that an increase in the expression of AGPATs is linked to barrier requirements.

The etiology of atherosclerosis is complex and multifactorial but there is extensive evidence indicating that oxidized lipoproteins may play a key role. At present, the site and mechanism by which lipoproteins are oxidized are not resolved, and it is not clear if oxidized lipoproteins form locally in the artery wall and/or are sequestered in atherosclerotic lesions following the uptake of circulating oxidized lipoproteins. We have been focusing our studies on demonstrating that such potentially atherogenic oxidized lipoproteins in the circulation are at least partially derived from oxidized lipids in the diet. Thus, the purpose of our work has been to determine in humans whether oxidized dietary oxidized fats such as oxidized fatty acids and oxidized cholesterol are absorbed and contribute to the pool of oxidized lipids in circulating lipoproteins. When a meal containing oxidized linoleic acid was fed to normal subjects, oxidized fatty acids were found only in the postprandial chylomicron/chylomicron remnants (CM/RM) which were cleared from circulation within 8 h. No oxidized fatty acids were detected in low density lipoprotein (LDL) or high density lipoprotein (HDL) fractions at any time. However, when alpha-epoxy cholesterol was fed to human subjects, alpha-epoxy cholesterol in serum was found in CM/RM and also in endogenous very low density lipoprotein, LDL, and HDL and remained in the circulation for 72 h. In vitro incubation of the CM/RM fraction containing alpha-epoxy cholesterol with human LDL and HDL that did not contain alpha-epoxy cholesterol resulted in a rapid transfer of oxidized cholesterol from CM/RM to both LDL and HDL. We have suggested that cholesteryl ester transfer protein is mediating the transfer. Thus, alpha-epoxy cholesterol in the diet is incorporated into CM/RM fraction and then transferred to LDL and HDL contributing to lipoprotein oxidation. We hypothesize that diet-derived oxidized fatty acids in chylomicron remnants and oxidized cholesterol in remnants and LDL accelerate atherosclerosis by increasing oxidized lipid levels in circulating LDL and chylomicron remnants. This hypothesis is supported by our feeding experiments in animals. When rabbits were fed oxidized fatty acids or oxidized cholesterol, the fatty streak lesions in the aorta were increased by 100%. Moreover, dietary oxidized cholesterol significantly increased aortic lesions in apo-E and LDL receptor-deficient mice. A typical Western diet is rich in oxidized fats and therefore could contribute to the increased arterial atherosclerosis in our population.