Biomedical subjects

Lee K Marles

Publications and source records attributed to Lee K Marles.

5 recordsLinked to original sources

PubMed2005

A novel mechanism in control of human pigmentation by {beta}-melanocyte-stimulating hormone and 7-tetrahydrobiopterin.

The human skin holds the full machinery for pro-opiomelanocortin processing. The alpha-melanocyte-stimulating hormone (alpha-MSH)/melanocortin-1-receptor cascade has been implicated as a major player via the cAMP signal in the control of melanogenesis. Only very recently the beta-endorphin/mu-opiate receptor signal has been added to the list of regulators of melanocyte dendricity and melanin formation. In this context it was reported that (6R)-l-erythro-5,6,7,8-tetrahydrobiopterin (6BH(4)) can act as an allosteric inhibitor of tyrosinase, the key enzyme in melanogenesis, and this inhibition is reversible by both alpha- and beta-MSH. It was also shown earlier that 7BH(4), the isomer of 6BH(4), is twice as active in this inhibition reaction. However, as yet it is not known whether 7BH(4) is indeed present in loco in the melanosome. We here provide evidence that this isomer is present in this organelle in a concentration range up to 50 x 10(-6) M. Determination of beta-MSH in melanosomal extracts yielded 10 pg/mg protein. Moreover, we demonstrate reactivation of the 7BH(4)/tyrosinase inhibitor complex by beta-MSH, whereas alpha-MSH failed to do so. Furthermore, we show intra-melanosomal l-dopa formation from dopachrome by 7BH(4) in a concentration range up to 134 x 10(-6) M. Based on these results, we propose a new receptor-independent mechanism in the control of tyrosinase/melanogenesis by beta-MSH and the pterin 7BH(4).

Animals↗

PubMed2004

Perturbed 6-tetrahydrobiopterin recycling via decreased dihydropteridine reductase in vitiligo: more evidence for H2O2 stress.

To date there is ample evidence that patients with vitiligo accumulate millimolar concentrations of hydrogen peroxide (H2O2) in their epidermis as well as in their blood lymphocytes/monocytes. Several enzymes are affected by this H2O2 including catalase, glutathione peroxidase, and 4 alpha-carbinolamine dehydratase. The latter enzyme disrupts the recycling of the essential cofactor (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (6BH4) for the aromatic amino acid hydroxylases as well as the nitric oxide synthases. In this report we have elucidated the influence of H2O2 on dihydropteridine reductase (DHPR), the last enzyme in the 6BH4-recycling process. Here we show for the first time that concentrations of less than 30 microM H2O2 increase DHPR activities, whereas levels greater than 30 microM H2O2 deactivate the enzyme based on the oxidation of Met146 and Met151 in the sequence, consequently leading to disruption of the NADH-dependent enzyme active site. This oxidation was confirmed by Fourier transform-Raman spectroscopy yielding the expected SO band at 1025 cm-1 characteristic of methionine sulfoxide. Hence these results unmasked a novel regulatory mechanism for DHPR enzyme activity. Moreover, we also demonstrated that DHPR activities in whole blood of patients with vitiligo are significantly decreased in untreated patients, whereas activities are normalized after removal of epidermal H2O2 with a topical pseudocatalase (PC-KUS). Taken together, these new data add more evidence to a systemic involvement of H2O2 in the pathomechanism of vitiligo.

Adolescent↗

PubMed2004

Autocrine catecholamine biosynthesis and the beta-adrenoceptor signal promote pigmentation in human epidermal melanocytes.

Earlier it has been shown that human proliferating/undifferentiated basal keratinocytes hold the full capacity for autocrine catecholamine synthesis/degradation and express beta2-adrenoceptors (beta2-AR). In this report, we show that human melanocytes also express all of the mRNA and enzymes for autocrine synthesis of norepinephrine but fail to produce epinephrine. So far, it was established that human melanocytes express alpha1-AR which are induced by norepinephrine yielding the inosine triphosphate diacylglycerol signal. The presence of catecholamine synthesis and the beta2-AR signal escaped definition at that time. Using RT-PCR, immunofluorescence and radioligand binding with the beta2-AR antagonist (-)-[3H]CGP 12177, we show here that human melanocytes express functional beta2-AR (4230 receptors per cell) with a Bmax at 129.3 and a KD of 3.19 nM but lack beta1-AR expression. beta2-AR stimulation with epinephrine 10(-6) M and salbutamol 10(-6)-10(-5) M yielded a strong cyclic adenosine monophospate (cAMP) response in association with upregulated melanin production. Taken together these results indicate that the biosynthesis and release of epinephrine (10(-6) M) by surrounding keratinocytes can provide the cAMP response leading to melanogenesis in melanocytes via the beta2-AR signal. Moreover, the discovery of this catecholaminergic cAMP response in melanocytes adds a new source for this important second messenger in melanogenesis.

Autocrine Communication↗

PubMed2003

Increased epidermal functioning wild-type p53 expression in vitiligo.

Despite the lack of protective melanin and increased oxidative stress due to mM concentrations of epidermal H2O2 in vitiligo, there is no significantly increased risk for chronic actinic damage and non-melanoma skin cancer. Therefore the question arises, which protective mechanisms could be involved in the skin of these patients preventing the initiation of these cancers. Recently an overexpression of p53 has been shown in vitiligo. Unfortunately there was no further characterization of this elevated p53. Employing a functional colour yeast assay, the study presented herein demonstrates for the first time the overexpression of a functioning wild-type p53 protein in both depigmented and 'normal' pigmented epidermis of patients with vitiligo compared with healthy controls. Surprisingly long-term narrowband UVB (311 nm) treatment does not alter this expression. Moreover, MDM-2, PCNA and p21 protein expression remain unchanged compared with healthy controls. This increased epidermal p53 in vitiligo coincides with decreased thioredoxin reductase (TR) protein levels in both depigmented and pigmented skin whereas mRNA expression is unaffected. Because TR is one transcriptional target of p53, these results support a wild-type functionality, which was further supported by the specific p53 FASAY yeast test. To our knowledge this is the first example of persistent elevated functioning wild-type p53 in humans. Based on our results we hypothesize that the low incidence for actinic damage, basal cell and squamous cell carcinoma as documented in vitiligo could well reside in a protective function of up-regulated wild-type p53.

Adolescent↗

PubMed2003

Tyrosine hydroxylase isoenzyme I is present in human melanosomes: a possible novel function in pigmentation.

Both human epidermal melanocytes and keratinocytes have the full capacity for de novo synthesis of 6(R) L-erythro 5,6,7,8, tetrahydrobiopterin, the essential cofactor for the rate limiting step in catecholamine synthesis, via tyrosine hydroxylase. Catecholamine synthesis has been demonstrated in proliferating keratinocytes of the epidermis in human skin. This study presented herein identified for the first time the expression of tyrosine hydroxylase isozyme I mRNA within the melanocyte. The location of the enzyme was demonstrated in both the cytosol and melanosomes of human epidermal melanocytes, using immunohistochemistry and immunofluorescence double staining as well as immunogold electron microscopy. High-performance liquid chromatography (HPLC) analysis of pure melanosomal extracts from the human melanoma cell line, FM94, confirmed the production of L-dopa within these organelles. In addition, enzyme activities for both tyrosine hydroxylase and tyrosinase were measured in the same preparations, by following the catalytic release of tritiated water from L-[3,5-3H]tyrosine. The melanosomal membrane location of tyrosine hydroxylase together with tyrosinase implies a coupled interaction, where L-dopa production facilitates the activation of tyrosinase. Our results support a direct function for tyrosine hydroxylase in the melanosome via a concerted action with tyrosinase to promote pigmentation.

Cells, Cultured↗