Morphological and transcriptional evaluation of multiple facial cutaneous hyperpigmented spots.

Background: Morphological characteristics of major facial hyperpigmented spots have been well documented. However, detailed alterations of respective transcriptional profile for each spot and in-depth comparisons across multiple spot types have not been reported. Objectives: To comprehensively assess and compare multiple facial hyperpigmented spot types at the morphological and molecular levels by utilising transcriptional expression profiling with correlation to quantified histological features. Methods: Multiple types of facial spot biopsies were collected from Chinese women and compared to additional biopsies taken from adjacent healthy skin. The types of spots included Solar Lentigos with both elongated dermal-epidermal junction (DEJ) (SL[E]) and flat DEJ (SL[F]), Seborrhoeic Keratosis (SK), Melasma, Freckles, Post-inflammatory hyperpigmentation of resolving acne (PIH[A]) and other stimuli (PIH[O]). Combined histomorphometry, immunohistology, and transcriptome analysis for suprabasal-epidermis, basal-epidermis, and dermal compartments dissected by Laser Capture Microdissection (LCM) were conducted and compared across different spot types. Results: Each spot type was confirmed to have the unique histological pathology already documented elsewhere. Most of the spot types except Melasma and PIH (A) revealed similar melanocyte density to adjacent skin. All spots exhibited increased melanin synthesis, melanosome transportation, as well as enhanced melanocyte dendricity, however, each spot revealed a distinct transcriptome regulation pattern in pigmentation pathways. Upregulation of pigmentation genes was also observed in the dermis of SL(F), SL(E), SK and PIH(O), associated with significant modulation of DEJ related genes in basal-epidermis and/or dermal compartments, suggesting potential melanocyte infiltration into the dermis due to impaired DEJ quality. Beyond upregulated pigmentation, for most spots, gene expression in the suprabasal-epidermis regulating keratinisation was significantly upregulated in conjunction with thickened stratum corneum. Furthermore, downregulation of tight junction related genes represented by claudin-1 was observed in majority of spot types, suggesting compromised barrier function could be a similarity across spots. Additionally, Cyclin-Dependent Kinase Inhibitor 2A (CDKN2A) was upregulated in all types of spots, indicating involvement of cell senescence as a common theme. Conclusion: This comprehensive and comparative study based on the histological and transcriptional analysis of three skin compartments provided unique insights into specific causations as well as differences and similarities across multiple hyperpigmented spot types.

A regulator of ubiquitin-proteasome activity, 2-hexyldecanol, suppresses melanin synthesis and the appearance of facial hyperpigmented spots.

BACKGROUND: 2-Hexyldecanol has long been used in skin-care products, but has not previously been reported as an active ingredient for skin benefits. OBJECTIVES: To evaluate 2-hexyldecanol in in vitro and ex vivo systems and, if found to be active, progress it to topical clinical testing to determine effects on pigmentation in skin. METHODS: 2-Hexyldecanol was tested in melanocyte cell culture systems (B16 mouse melanoma cells and normal human melanocytes) for its effect on proteolytic activity and melanin production, in the absence and presence of the proteasome-specific inhibitor, MG132. It was further tested in a human skin explant model for its effect on melanin production. Lastly, topically applied 2-hexyldecanol was evaluated for its effect on the appearance of facial pigmentation in an 8-week, randomized, double-blind, vehicle-controlled, split-face incomplete block design study in Chinese women. RESULTS: In submerged cell culture, 2-hexyldecanol upregulated proteolytic activity and decreased melanin synthesis. These effects were antagonized by the proteasome-specific inhibitor MG132. MG132, tested in the absence of 2-hexyldecanol, increased melanin production. In a human skin explant model, topical 2-hexyldecanol suppressed the production of melanin vs. a vehicle control. In a human clinical study in Chinese women (n = 110 observations per test material), a 2-hexyldecanol-containing formulation significantly reduced the appearance of facial hyperpigmented spots vs. its control. CONCLUSIONS: These data indicate that regulation of proteasome activity is a viable target for control of melanin production, that 2-hexyldecanol upregulates proteasomal activity in melanocytes, and that topical 2-hexyldecanol reduces the appearance of hyperpigmentation.

alpha 2A/alpha 2C-adrenergic receptor third loop chimera show that agonist interaction with receptor subtype backbone establishes G protein-coupled receptor kinase phosphorylation.

The alpha(2A)-adrenergic receptor (AR) undergoes rapid agonist-promoted desensitization due to phosphorylation by G protein-coupled receptor kinases (GRKs) 2 and 3 at serines in the third intracellular loop of the receptor. In contrast, the alpha(2C)AR fails to display such desensitization or phosphorylation, which has been presumed to be due to this receptor lacking GRK phosphorylation sites. However, the alpha(2C)AR has multiple serines and threonines in putative favorable motifs within its third intracellular loop. We considered that the conformation of the third intracellular loop imposed by agonists binding to the transmembrane-spanning domains could be the basis of this subtype-specific property, rather than the presence or absence of phosphoacceptors per se. To address this, alpha(2A)/alpha(2C) third loop chimeric receptors were constructed. In whole cell phosphorylation studies, the alpha(2A) with the alpha(2C) third loop receptor underwent agonist-promoted phosphorylation while the alpha(2C) with the alpha(2A) third loop receptor did not, indicating that the agonist interaction with the parent receptor backbone establishes the phosphorylation phenotype. We postulated then that agonists with diverse structures that distinctly interact with alpha(2)AR should display different degrees of phosphorylation independent of receptor activation. Indeed, several full and partial agonists were identified, which evoked phosphorylation that was not related to intrinsic activity as established by [(35)S]guanosine 5'-3-O-(thio)triphosphate binding. Taken together, it appears that phosphorylation of the alpha(2)AR evoked by agonist is highly sensitive to the conformation of the third intracellular loop induced/stabilized by agonist to such an extent that these properties dictate the extent of phosphorylation of the loop when phosphoacceptors are present, and are the basis for subtype-specific phosphorylation.

Agonist-mediated downregulation of G alpha i via the alpha 2-adrenergic receptor is targeted by receptor-Gi interaction and is independent of receptor signaling and regulation.

One mechanism of long-term agonist-promoted desensitization of alpha2AR function is downregulation of the cellular levels of the alpha subunit of the inhibitory G protein, Gi. In transfected CHO cells expressing the human alpha2AAR, a 40.1 +/- 3.3% downregulation of Galphai2 protein occurred after 24 h of exposure of the cells to epinephrine, which was not accompanied by a decrease in Galphai2 mRNA. The essential step that targets Gi for degradation by agonist occupancy of the receptor was explored using mutated alpha2AAR lacking specific structural or functional elements. These consisted of 5HT1A receptor and beta2AR sequences substituted at residues 113-149 of the second intracellular loop and 218-235 and 355-371 of the N- and C-terminal regions of the third intracellular loop (altered Gi and Gs coupling), deletion of Ser296-299 (absent GRK phosphorylation), and substitution of Cys442 (absent palmitoylation and receptor downregulation). Of these mutants, only those with diminished Gi coupling displayed a loss of agonist-promoted Gi downregulation, thus excluding Gs coupling and receptor downregulation, palmitoylation, and phosphorylation as necessary events. Furthermore, coupling-impaired receptors consisting of mutations in the second or third loops ablated Gi downregulation, suggesting that a discreet structural motif of the receptor is unlikely to represent a key element in the process. While pertussis toxin ablated Gi downregulation, blocking downstream intracellular consequences of alpha2AAR activation or mimicking these pathways by heterologous means failed to implicate cAMP/adenylyl cyclase, phospholipase C, phospholipase D, or MAP kinase pathways in alpha2AAR-mediated Gi downregulation. Taken together, agonist-promoted Gi downregulation requires physical alpha2AAR-Gi interaction which targets Gi for degradation in a manner that is independent of alpha2AAR trafficking, regulation, or second messengers.

Phosphorylation and functional desensitization of the alpha2A-adrenergic receptor by protein kinase C.

We have investigated the potential for protein kinase C (PKC) to phosphorylate and desensitize the alpha2A-adrenergic receptor (alpha2AAR). In whole-cell phosphorylation studies, recombinantly expressed human alpha2AAR displayed an increase in phosphorylation after short-term exposure to 100 nM phorbol 12-myristate-13-acetate (PMA) that was blocked by preincubation with a PKC inhibitor. This increase in receptor phosphorylation over basal amounted to 172 +/- 40% in COS-7 cells and 201 +/- 40% in Chinese hamster ovary cells. In permanently transfected Chinese hamster fibroblast cells, PKC activation by brief exposure of the cells to PMA resulted in a marked desensitization of alpha2AAR function, amounting to a 68 +/- 4% decrease in the maximal agonist (UK14304)-stimulated intracellular calcium release. Such desensitization was blocked by the PKC inhibitor bisindolylmaleimide I and was not evoked by an inactive phorbol ester. The desensitization of this agonist response was not caused by PKC-mediated augmentation of G protein-coupled receptor kinase activity, because PMA-promoted desensitization of a mutated alpha2AAR that lacked G protein-coupled receptor kinase phosphorylation sites was identical to that of wild-type alpha2AAR. To test whether PKC phosphorylation is a mechanism by which alpha2AAR can be regulated by other receptors, the alpha1bAR was co-expressed with the alpha2AAR in Chinese hamster ovary cells. Upon selective activation of alpha1bAR, the function of alpha2AAR underwent a 53 +/- 5% desensitization. Thus, cellular events that result in PKC activation promote phosphorylation of the alpha2AAR and lead to substantial desensitization of receptor function. This heterologous regulation also represents a mechanism by which rapid crosstalk between the alpha2AAR and other receptors can occur.

Role of the amino terminus of the third intracellular loop in agonist-promoted downregulation of the alpha2A-adrenergic receptor.

A prominent feature of long-term regulation of the alpha2A-adrenergic receptor (alpha2AAR) is a loss of cellular receptors over time (downregulation). The molecular determinants of downregulation were sought by targeting regions of the receptor involved in G protein coupling and phosphorylation. Mutated receptors, consisting of chimeric substitutions of analogous beta2-adrenergic receptor (beta2AR) and serotonin 5-hydroxytryptamine1A (5-HT1A) receptor sequence into the second intracellular loop (ICL2) (residues 113-149), the amino terminus (residues 218-235) and carboxy terminus (residues 355-371) of ICL3, and a deletion of the beta-adrenergic receptor kinase (betaARK) phosphorylation sites in the third intracellular loop (ICL3) (residues 293-304), were expressed in Chinese hamster ovary (CHO) cells. Wild-type alpha2AAR underwent 31% +/- 3% downregulation after 24 h of exposure to 100 microM epinephrine. Loss of downregulation was observed with some mutants, but this was not related to functional coupling to inhibitory or stimulatory guanine nucleotide regulatory binding proteins (Gi or GS) or to phosphorylation. Rather, any mutant with a substitution of the amino terminus of ICL3 (regardless of whether the substitution was with beta2AR or 5-HT1A sequence) resulted in upregulation. Studies with an inhibitor of protein synthesis indicated that the primary mechanism of downregulation of the alpha2AAR is agonist-promoted degradation of receptor protein which requires a destabilization sequence in the amino terminus of ICL3. Thus, in contrast to other G protein-coupled receptors, in which G protein coupling or phosphorylation are critical for long-term agonist regulation, the alpha2AAR has a specific structural domain distinct from these other functional regions that serves to direct agonist-promoted downregulation.

Mutational analysis of Glu-327 of Na(+)-K(+)-ATPase reveals stimulation of 86Rb+ uptake by external K+.

A competition assay of 86Rb+ uptake in HeLa cells transfected with ouabain-resistant Na(+)-K(+)-ATPase mutants revealed a stimulation of 86Rb+ uptake at low external concentrations (1 mM) of competitor (K+). Of the models that were tested, those that require that two K+ be bound before transport occurs gave the worst fits. Random and ordered binding schemes described the data equally well. General models in which both binding and transport were allowed to be cooperative yielded parameter errors larger than the parameters themselves and could not be utilized. Models that assumed noncooperative transport always showed positive cooperativity in binding. E327Q and E327L mutated forms of rat alpha 2 had lower apparent affinities for the first K+ bound than did wild-type rat alpha 2 modified to be ouabain resistant. The mutations did not affect the apparent affinity of the second K+ bound. Models that assumed noncooperativity in binding always showed positively cooperative transport, i.e., enzymes with two K+ bound had a higher flux than those with one K+ bound. Increases in external Na+ decreased the apparent affinity for K+ for all models and decreased the ratio of the apparent influx rate constants for E327L.

G protein-coupled receptor kinase specificity for phosphorylation and desensitization of alpha2-adrenergic receptor subtypes.

The alpha2-adrenergic receptor (alpha2AR) subtype alpha2C10 undergoes rapid agonist-promoted desensitization which is due to phosphorylation of the receptor. One kinase that has been shown to phosphorylate alpha2C10 in an agonist-dependent manner is the betaAR kinase (betaARK), a member of the family of G protein-coupled receptor kinases (GRKs). In contrast, the alpha2C4 subtype has not been observed to undergo agonist-promoted desensitization or phosphorylation by betaARK. However, the substrate specificities of the GRKs for phosphorylating alpha2AR subtypes are not known. We considered that differential capacities of various GRKs to phosphorylate alpha2C10 and alpha2C4 might be a key factor in dictating in a given cell the presence or extent of agonist-promoted desensitization of these receptors. COS-7 cells were co-transfected with alpha2C10 or alpha2C4 without or with the following GRKs: betaARK, betaARK2, GRK5, or GRK6. Intact cell phosphorylation studies were carried out by labeling cells with 32Pi, exposing some to agonist, and purifying the alpha2AR by immunoprecipitation and SDS-polyacrylamide gel electrophoresis. BetaARK and betaARK2 were both found to phosphorylate alpha2C10 to equal extents (>2-fold over that of the endogenous kinases). On the other hand, GRK5 and GRK6 did not phosphorylate alpha2C10. In contrast to the findings with alpha2C10, alpha2C4 was not phosphorylated by any of these kinases. Functional studies carried out in transfected HEK293 cells expressing alpha2C10 or alpha2C4 and selected GRKs were consistent with these phosphorylation results. With the marked expression of these receptors, no agonist-promoted desensitization was observed in the absence of GRK co-expression. However, desensitization was imparted to alpha2C10 by co-expression of betaARK but not GRK6, while alpha2C4 failed to desensitize with co-expression of betaARK. These results indicate that short term agonist-promoted desensitization of alpha2ARs by phosphorylation is dependent on both the receptor subtype and the expressed GRK isoform.

An acidic motif within the third intracellular loop of the alpha2C2 adrenergic receptor is required for agonist-promoted phosphorylation and desensitization.

The alpha 2C2 adrenergic receptor contains a highly acidic stretch of amino acids (EDEAEEEEEEEEEEEE) within the third intracellular loop. To investigate the role of this region, we utilized site-directed mutagenesis to delete these 16 amino acids as well as to substitute them with glutamine, thereby conserving size but not charge. The wild-type and mutated alpha 2C2 receptors were permanently expressed in CHO cells. Neither substitution nor deletion of this region affected receptor expression, agonist or antagonist binding affinities, guanine nucleotide-sensitive formation of the high-affinity agonist-receptor-G protein complex, or functional coupling of the receptor to Gi. We considered that since alpha 2C2 agonist-promoted desensitization is due to phosphorylation by the beta-adrenergic receptor kinase (or a related kinase), that this region may be important for establishing the acidic mileau required by this kinase. Therefore, the consequences of 30 min of agonist preexposure on subsequent alpha 2C2-mediated inhibition of adenylyl cyclase and on high-affinity agonist binding were determined for the wild-type and these two mutants. The wild-type alpha 2C2 receptor underwent approximately 52% functional desensitization and a approximately 40% loss of high-affinity binding after such exposure. In contrast, deletion and substitution of this acidic stretch of amino acids ablated desensitization as assessed by both approaches. These results correlated with those obtained in whole cell phosphorylation experiments. Cells expressing each receptor were incubated with [32P]orthophosphate and exposed to agonist, and receptors were purified by immunoprecipitation. The deletion and the substitution mutant receptors underwent agonist-promoted phosphorylation at levels of only 44 +/- 5% and 50 +/- 15%, respectively, relative to wild-type alpha 2C2.(ABSTRACT TRUNCATED AT 250 WORDS)

Tissue- and isoform-specific kinetic behavior of the Na,K-ATPase.

The objective of this study has been to delineate the side-specific effects of Na+ and K+ on the transport kinetics of tissue-specific Na/K pumps. Two experimental systems have been used. In one, Na/K pumps of exogenous microsomal membrane sources (rat axolemma, kidney) were delivered by membrane fusion into dog erythrocytes, and in the other, the three isoforms of the catalytic subunit of the rat enzyme were individually transfected into HeLa cells as in previous studies (Jewell, E.A., and Lingrel, J. B (1991) J. Biol. Chem. 266, 16925-16930), with the alpha 2 and alpha 3 isoforms rendered relatively resistant to ouabain by site-directed mutagenesis. Whereas the kidney microsomes comprise the alpha 1 catalytic isoform, the axolemma microsomes were predominantly alpha 3 (approximately 60%) with lesser amounts of alpha 2 (approximately 25%) and alpha 1 (approximately 15%) as measured by the ouabain-sensitive profile of phosphoenzyme as well as by immunoblotting with isoform-specific antibodies using membranes of known specific activity as standards (alpha 1 of kidney, alpha 1 and alpha 2 of muscle). Both systems were analyzed with respect to the effects of varying concentrations of cytoplasmic Na+ and extracellular K+ on pump-mediated 86Rb+(K+) influx. With the individual isoform-transfected HeLa cells and monensin added to vary and control the intracellular Na+ concentration, differences in apparent affinities of the alpha 3 isoform compared with the alpha 1 and alpha 2 isoforms were observed, i.e. a approximately 3-fold higher affinity for extracellular K+ and approximately 4-fold lower affinity for cytoplasmic Na+. Thus, in the presence of 10 mM extracellular Na+, apparent K0.5 values for extracellular K+ activation of K+(Rb+) influxes were 0.22 +/- 0.02 mM for alpha 1, 0.20 +/- 0.02 mM for alpha 2, and 0.09 +/- 0.01 mM for alpha 3. At high intracellular K+ (> or = 100 mM) and saturating extracellular K+ concentrations, apparent K0.5 values for cytoplasmic Na+ activation were 17.6 +/- 1.1 mM for alpha 1, 19.7 +/- 1.0 mM for alpha 2, and 63.5 +/- 9.1 mM for alpha 3. The functional differences observed with the individual isoform-transfected cells were completely consistent with the kinetic differences observed with the axolemma and kidney pumps fused into erythrocytes. Axolemma pumps had a approximately 3-fold lower K0.5 for extracellular K+ and a approximately 2-fold higher K0.5 for cytoplasmic Na+.(ABSTRACT TRUNCATED AT 400 WORDS)

Structure-function studies of the Na,K-ATPase.

Na,K-ATPase catalyzes the movement of sodium and potassium ions across the cell membrane utilizing ATP as an energy source. This enzyme is present in almost all tissues of higher organisms but is most abundant in the kidney where it is responsible for reabsorbing sodium ions from the glomerular filtrate. The enzyme is composed of two subunits and serves as the receptor for cardiac glycosides. Utilizing an expression/selection system it has been possible to identify several amino acid residues that affect sensitivity to the cardiac glycoside, ouabain. Those identified to date are located in the first transmembrane region and first extracellular region. The fact that amino acid residues within a transmembrane region affect ouabain sensitivity suggests that the drug is partially internalized in the lipid bilayer. In an effort to determine whether any of the amino acid residues which affect ouabain sensitivity interact with the sugar part of cardiac glycosides, ouabain and ouabagenin were tested in terms of their ability to inhibit enzyme containing substitutions at these positions. The two compounds differ in that ouabagenin lacks a sugar moiety. Interestingly, the ratio of I50's for the substituted enzymes remains the same as the wild type, which suggests that the amino acids identified as determinants of ouabain sensitivity to date are not likely to interact with the sugar. Another set of studies focused on cation binding. It has been proposed that cation transport involves negatively charged residues in one or more transmembrane regions.(ABSTRACT TRUNCATED AT 250 WORDS)

Site-directed mutagenesis of the Na,K-ATPase: consequences of substitutions of negatively-charged amino acids localized in the transmembrane domains.

Site-directed mutagenesis was used to examine the importance of five carboxyl-containing amino acids localized in the putative membrane-spanning regions of the Na,K-ATPase (i.e., E327, E778, D803, D807, and D925 of the rat alpha 2 isoform). The substitutions were introduced into a cDNA encoding a ouabain-resistant isoform (i.e., rat alpha 2* which was mutated to encode a ouabain-resistant isoform), and the effect of these substitutions on Na,K-ATPase function was assessed by screening the altered enzymes for their ability to confer ouabain resistance when expressed in otherwise ouabain-sensitive cells. The expression of the alpha isoform containing certain substitutions at positions 327 and 925 was able to confer ouabain resistance to HeLa cells while the expression of rat alpha 2* containing substitutions at positions 778, 803, and 807 was not. In particular, amino acids in each of these positions were substituted with leucine to evaluate the importance of the carboxyl-containing side chain. The ability of rat alpha 2* containing E327L and D925L to confer ouabain resistance to HeLa cells indicates that neither the negative charge nor the oxygen-containing side chain is absolutely essential for overall function in this position. In contrast, the inability of rat alpha 2* carrying E778L, D803L, and D807L to confer ouabain resistance suggests that the naturally occurring amino acid may be more critical structurally and/or functionally for the Na,K-ATPase. Other more conservative substitutions introduced to further characterize the role of particular amino acid side chains include E327D, E327Q, D803N, D803E, and D925N.(ABSTRACT TRUNCATED AT 250 WORDS)