In vitro culture of fibroblasts from pubic skin of women in diagnosis of androgen hypersensitivity]

Intracellular metabolism of testosterone was studied in in vitro cultured skin fibroblasts obtained from women with idiopathic hirsutism. A significantly higher rate of conversion of testosterone into 5-alpha-dihydrotestosterone was found in these women (4.78 +/- 2.08 fmol/microgram DNA/hour) as compared to that obtained for skin cultures of women without symptoms of androgenization (0.98 +/- 0.37 fmol/microgram DNA/hour). These results demonstrate that fibroblast culture may be useful in diagnosing the causes of hyperandrogenization in women with normal androgen secretion.     

A novel SOS1 mutation in Costello/CFC syndrome affects signaling in both RAS and PI3K pathways

Abstract

Context: Pathological upregulation of the RAS/MAPK pathway causes Costello, Noonan and cardio–facio–cutaneous (CFC) syndrome; however, little is known about PI3K/AKT signal transduction in these syndromes. Previously, we found a novel mutation of the SOS1 gene (T158A) in a patient with Costello/CFC overlapping phenotype. Objective: The aim of this study was to investigate how this mutation affects RAS/MAPK as well as PI3K/AKT pathway signal transduction.

Materials and methods: Wild-type and mutant (T158A) Son of Sevenless 1 (SOS1) were transfected into 293T cells. The levels of phospho- and total ERK1/2, AKT, p70S6K and pS6 were examined under epidermal growth factor (EGF) stimulation. Results: After EGF stimulation, the ratio of phospho-ERK1/2 to total ERK1/2 was highest at 5?min in mutant (T158A) SOS1 cells, and at 15?min in wild-type SOS1 cells. Phospho-AKT was less abundant at 60?min in mutant than in wild-type SOS1 cells. Phosphorylation at various sites in p70S6K differed between wild-type and mutant cells. Eighteen hours after activation by EGF, the ratio of phospho-ERK1/2 to total ERK1/2 remained significantly higher in mutant than in wild-type SOS1 cells, but that of phospho-AKT to total AKT was unchanged. Discussion: T158A is located in the histone-like domain, which may have a role in auto-inhibition of RAS exchanger activity of SOS1. T158A may disrupt auto-inhibition and enhance RAS signaling. T158A also affects PI3K/AKT signaling, probably via negative feedback via phospho-p70S6K. Conclusion: The SOS1 T158A mutation altered the phosphorylation of gene products involved in both RAS/MAPK and PI3K/AKT pathways.     

Antihirsutism activity of Fennel (fruits of Foeniculum vulgare) extract. A double-blind placebo controlled study.

Idiopathic hirsutism is defined as the occurrence of excessive male pattern hair growth in women who have a normal ovulatory menstrual cycle and normal levels of serum androgens. It may be a disorder of peripheral androgen metabolism. In this study we evaluated the clinical response of idiopathic hirsutism to topical Fennel extract. Fennel, Foeniculum vulgare, is a plant, which has been used as an estrogenic agent. The ethanolic extract of Fennel was obtained by using a soxhlete apparatus. In a double blind study, 38 patients were treated with creams containing 1%, 2% of Fennel extract and placebo. Hair diameter was measured and rate of growth was considered. The efficacy of treatment with the cream containing 2% Fennel is better than the cream containing 1% Fennel and these two were more potent than placebo. The mean values of hair diameter reduction was 7.8%, 18.3% and -0.5% for patients receiving the creams containing 1%, 2% and 0% (placebo) respectively.     

Calcium- and salt-stress signaling in plants: shedding light on SOS pathway

As salt stress imposes a major environmental threat to agriculture, understanding the basic physiology and genetics of cell under salt stress is crucial for developing any transgenic strategy. Salt Overly Sensitive (SOS) genes (SOS1-SOS3) were isolated through positional cloning. Since sos mutants are hypersensitive to salt, their characterization resulted in the discovery of a novel pathway, which has helped in our understanding the mechanism of salt-stress tolerance in plants. Genetic analysis confirmed that SOS1-SOS3 function in a common pathway of salt tolerance. This pathway also emphasizes the significance of Ca²⁺ signal in reinstating cellular ion homeostasis. SOS3, a Ca²⁺ sensor, transduces the signal downstream after activating and interacting with SOS2 protein kinase. This SOS3-SOS2 complex activates the Na⁺/H⁺ antiporter activity of SOS1 thereby reestablish cellular ion homeostasis. Recently, SOS4 and SOS5 have also been characterized. SOS4 encodes a pyridoxal (PL) kinase that is involved in the biosynthesis of pyridoxal-5-phosphate (PLP), an active form of vitamin B6. SOS5 has been shown to be a putative cell surface adhesion protein that is required for normal cell expansion. Under salt stress, the normal growth and expansion of a plant cell becomes even more important and SOS5 helps in the maintenance of cell wall integrity and architecture. In this review we focus on the recent advances in salt stress and SOS signaling pathway. A broad coverage of the discovery of SOS mutants, structural aspect of these genes and the latest developments in the field of SOS1-SOS5 has been described.     

Salmon nasal cartilage proteoglycan enhances growth of normal human dermal fibroblast through Erk1/2 phosphorylation

Proteoglycan (PG) is a heavily glycosylated protein, localized to cell surface and extracellular matrix, and has various functions. Recently, it has been gradually revealed that PG interacts with various growth factors and morphogens and regulates cellular functions. Although salmon nasal cartilage PG (Salmon-PG) increases proliferation of immortalized cells, its mechanism remains unclear. In this study, we confirmed the effect of Salmon-PG on normal human dermal fibroblast (NHDF) and investigated the mechanism of PG action on NHDF. Salmon-PG dose- and time-dependently increased NHDF proliferation. Receptor tyrosine kinase array revealed that Salmon-PG increased only Erk1/2 signaling. Erk1/2 phosphorylation was significantly increased by Salmon-PG in a time-(10 min) and dose-(400 or 800 μg/mL) dependent manner. MEK inhibitor suppressed the enhancement of NHDF proliferation by Salmon-PG. The overall findings indicate that Salmon-PG plays a role as a growth factor in NHDF via Erk1/2 activation, suggesting that Salmon-PG contributes to the maintenance of skin homeostasis.     

Regulation of vacuolar Na+/H+ exchange in Arabidopsis thaliana by the salt-overly-sensitive (SOS) pathway

For plants growing in highly saline environments, accumulation of sodium in the cell cytoplasm leads to disruption of metabolic processes and reduced growth. Maintaining low levels of cytoplasmic sodium requires the coordinate regulation of transport proteins on numerous cellular membranes. Our previous studies have linked components of the Salt-Overly-Sensitive pathway (SOS1-3) to salt tolerance in Arabidopsis thaliana and demonstrated that the activity of the plasma membrane Na+/H+ exchanger (SOS1) is regulated by SOS2 (a protein kinase) and SOS3 (a calcium-binding protein). Current studies were undertaken to determine if the Na+/H+ exchanger in the vacuolar membrane (tonoplast) of Arabidopsis is also a target for the SOS regulatory pathway. Characterization of tonoplast Na+/H+ exchange demonstrated that it represents activity originating from the AtNHX proteins since it could be inhibited by 5-(N-methyl-N-isobutyl)amiloride and by anti-NHX1 antibodies. Transport activity was selective for sodium (apparent Km=31 mm) and electroneutral (one sodium ion for each proton). When compared with tonoplast Na+/H+-exchange activity in wild type, activity was significantly higher, greatly reduced, and unchanged in sos1, sos2, and sos3, respectively. Activated SOS2 protein added in vitro increased tonoplast Na+/H+-exchange activity in vesicles isolated from sos2 but did not have any effect on activity in vesicles isolated from wild type, sos1, or sos3. These results demonstrate that (i) the tonoplast Na+/H+ exchanger in Arabidopsis is a target of the SOS regulatory pathway, (ii) there are branches to the SOS pathway, and (iii) there may be coordinate regulation of the exchangers in the tonoplast and plasma membrane.     

Sexual hormones in human skin.

The skin locally synthesizes significant amounts of sexual hormones with intracrine or paracrine actions. The local level of each sexual steroid depends upon the expression of each of the androgen- and estrogen-synthesizing enzymes in each cell type, with sebaceous glands and sweat glands being the major contributors. Sebocytes express very little of the key enzyme, cytochrome P450c17, necessary for synthesis of the androgenic prohormones dehydroepiandrosterone and androstenedione, however, these prohormones can be converted by sebocytes and sweat glands, and probably also by dermal papilla cells, into more potent androgens like testosterone and dihydrotestosterone. Five major enzymes are involved in the activation and deactivation of androgens in skin. Androgens affect several functions of human skin, such as sebaceous gland growth and differentiation, hair growth, epidermal barrier homeostasis and wound healing. Their effects are mediated by binding to the nuclear androgen receptor. Changes of isoenzyme and/or androgen receptor levels may have important implications in the development of hyperandrogenism and the associated skin diseases such as acne, seborrhoea, hirsutism and androgenetic alopecia. On the other hand, estrogens have been implicated in skin aging, pigmentation, hair growth, sebum production and skin cancer. Estrogens exert their actions through intracellular receptors or via cell surface receptors, which activate specific second messenger signaling pathways. Recent studies suggest specific site-related distribution of ERalpha and ERbeta in human skin. In contrast, progestins play no role in the pathogenesis of skin disorders. However, they play a major role in the treatment of hirsutism and acne vulgaris, where they are prescribed as components of estrogen-progestin combination pills and as anti-androgens. These combinations enhance gonadotropin suppression of ovarian androgen production. Estrogen-progestin treatment can reduce the need for shaving by half and arrest progression of hirsutism of various etiologies, but do not necessarily reverse it. However, they reliably reduce acne. Cyproterone acetate and spironolactone are similarly effective as anti-androgens in reducing hirsutism, although there is wide variability in individual responses.     

The Hirsute Woman: Challenges in Evaluation and Management

ObjectiveTo review the etiology, pathogenesis, diagnostic approach, and management of hirsutism.MethodsWe discuss the clinical course of hirsutism and provide our recommendations on the various treatment options available.ResultsHirsutism is a common clinical problem characterized by the presence of increased terminal hair growth in androgen-dependent areas of the skin. The development of hirsutism depends on the presence of the pilosebaceous unit, which is genetically determined, as well as the presence of the androgen receptor and intracellular 5α-reductase activity, which converts testosterone to its more active metabolite, dihydrotestosterone. A detailed history and physical examination and the following laboratory tests can diagnose most causes of hirsutism: early-morning follicular phase measurement of total testosterone, testosterone not bound to sex hormone-binding globulin, dehydroepiandrosterone sulfate, 17-hydroxypro- gesterone, prolactin, and thyrotropin levels. Oral contraceptive preparations may be effective monotherapy formild hirsutism. For the treatment of more severe hirsutism, oral contraceptive pills combined with spironolactone are as effective as oral contraceptive pills containing cyproter- one acetate, which are not available in the United States. Because of teratogenicity, spironolactone should be used with caution in premenopausal women when it is administered without an oral contraceptive pill. Metformin is an alternative therapy for hirsutism in women with polycystic ovary syndrome who have other indications for metformin use. Metformin is not as effective as antiandrogens for the management of hirsutism. The use of glucocorticoids, finasteride, or flutamide is not recommended.ConclusionsHirsutism can be evaluated with a detailed history and physical examination and a limited number of hormonal tests. Serious disorders presenting as hirsutism are rare and can be excluded with the recommended evaluation. Treatment is targeted at reducing the production and bioavailability of testosterone, as well as blocking target tissue androgen action. (Endocr Pract. 2011;17:807-818)     

A mutation in the SOS1 gene causes hereditary gingival fibromatosis type 1

Hereditary gingival fibromatosis (HGF) is a rare, autosomal dominant form of gingival overgrowth. Affected individuals have a benign, slowly progressive, nonhemorrhagic, fibrous enlargement of the oral masticatory mucosa. Genetic loci for autosomal dominant forms of HGF have been localized to chromosome 2p21-p22 (HGF1) and chromosome 5q13-q22 (HGF2). To identify the gene responsible for HGF1, we extended genetic linkage studies to refine the chromosome 2p21-p22 candidate interval to approximately 2.3 Mb. Development of an integrated physical and genetic map of the interval identified 16 genes. Sequencing of these genes, in affected and unaffected HGF1 family members, identified a mutation in the Son of sevenless-1 (SOS1) gene in affected individuals. In this report, we describe the genomic structure of the SOS1 gene and present evidence that insertion of a cytosine between nucleotides 126,142 and 126,143 in codon 1083 of the SOS1 gene is responsible for HGF1. This insertion mutation, which segregates in a dominant manner over four generations, introduces a frameshift and creates a premature stop codon, abolishing four functionally important proline-rich SH3 binding domains normally present in the carboxyl-terminal region of the SOS1 protein. The resultant protein chimera contains the wild-type SOS1 protein for the N-terminal amino acids 1-1083 fused to a novel 22-amino acid carboxyl terminus. Similar SOS1 deletion constructs are functional in animal models, and a transgenic mouse construct with a comparable SOS1 chimera produces a phenotype with skin hypertrophy. Clarification of the functional role of this SOS1 mutant has implications for understanding other forms of gingival fibromatosis and corrective gingival-tissue management.     

Urinary testosterone and epitestosterone excretions in women with idiopathic hirsutism.

Urinary testosterone and epitestosterone were determined in 90 normal healthy women and in 90 women with idiopathic hirsutism, both groups aged between 16 and 46 years. Testosterone and epitestosterone excretion values were above the normal range in 27 of the 90 hirsute women (30%), and these 27 women had much more prominent hair growth than the others. When these results were statistically analysed according to the age groups or for all ages as a whole, they were found to be highly significant (P less than 0.0005). Therefore, it is concluded that the estimation of urinary testosterone and epitestosterone could be meaningfully applied to study the androgen status of hirsute women.     

Reactive oxygen species mediate Na+-induced SOS1 mRNA stability in Arabidopsis.

Salt Overly Sensitive 1 (SOS1), a plasma membrane Na+/H+ antiporter in Arabidopsis, is a salt tolerance determinant crucial for the maintenance of ion homeostasis in saline stress conditions. SOS1 mRNA is unstable at normal growth conditions, but its stability is substantially increased under salt stress and other ionic and dehydration stresses. In addition, H2O2 treatment increases the stability of SOS1 mRNA. SOS1 mRNA is inherently unstable and rapidly degraded with a half-life of approximately 10 min. Rapid decay of SOS1 mRNA requires new protein synthesis. Stress-induced SOS1 mRNA stability is mediated by reactive oxygen species (ROS). NADPH oxidase is also involved in the upregulation of SOS1 mRNA stability, presumably through the control of extracellular ROS production. The cis-element required for SOS1 mRNA instability resides in the 500-bp region within the 2.2 kb at the 3' end of the SOS1 mRNA. Furthermore, mutations in the SOS1 gene render sos1 mutants more tolerant to paraquat, a non-selective herbicide causing oxidative stress, indicating that SOS1 plays negative roles in tolerance of oxidative stress. A hypothetical model for the signaling pathway involving SOS1-mediated pH changes, NADPH oxidase activation, apoplastic ROS production and downstream signaling transduction is proposed, and the biological significance of ROS-mediated induction of SOS1 mRNA stability is discussed.     

Molecular genetic and endocrine mechanisms of hair growth

The prenatal morphogenesis of hair follicles depends upon a precisely regulated series of molecular genetic processes. Hormones and their receptors play prominent roles in modulating postnatal hair cycling, which recapitulates some aspects of morphogenesis. The responses to androgen are the most obvious of these. The postnatal androgen sensitivity of pilosebaceous units in different skin areas is programmed during prenatal development to permit clinical outcomes such as hirsutism and pattern baldness. Thyroid hormone, glucocorticoids, insulin-like growth factor-I, and prolactin have clinically significant effects on specific aspects of hair growth. The nuclear receptors vitamin D receptor and retinoid X receptor are essential for postnatal hair cycling. Other hormones have less clear effects on hair growth. Advances in research on the interaction of hormone target genes with the biological processes involved in hair morphogenesis and cycling can be expected to improve management of hirsutism and alopecia.     

Association of insulin resistance with hyperandrogenia in women

In humans, the skin is a target tissue for androgen action; hair growth and sebum secretion are under active androgen control. An increased production or metabolism of testosterone, the main active androgen, shows up clinically in dermatological symptoms such as hirsutism, hyperseborrheic acne and alopecia. Polycystic ovary syndrome (PCOS) is the most frequent androgen disorder of ovarian function. PCOS patients have amenorrhea or severe oligomenorrhea, increased testosterone levels and most often enlarged polycystic ovaries on ultrasound examination. In addition, many PCOS patients have a tendency to accumulate abdominal fat and/or to develop obesity. Some also display a particular metabolic pattern including an atherogenic lipid profile, glucose intolerance and an increased fasting insulin level, which is known to be closely linked with an insulin resistant state. Several studies have now reported that PCOS patients show increased incidence of type 2 diabetes and cardiovascular disease. In addition to being a target for androgens the skin has abundant insulin receptors on the keratinocyte surface membrane and acanthosis nigricans is a common symptom of severe insulin resistance among patients with insulin receptor disorders. However, acanthosis nigricans could also be present in PCOS women given evidence of the intensity of their insulin resistance. This presentation will review the mutual relationship between hyperandrogenia and insulin resistance, with particular attention paid to: (1) insulin secretion and insulin sensitivity in PCOS; (2) the complexity of the molecular mechanisms involved in insulin resistance; (3) the paradoxical relationship between insulin resistance and hyperandrogenia; (4) the current genetic studies; and (5) new avenues for long-term treatment of PCOS women.     

Direct interaction of SOS1 Ras exchange protein with the SH3 domain of phospholipase C-gamma1

A recent report that microinjection of the SH3 domain of PLC-gamma1 could induce DNA synthesis raised the functional importance of the SH3 domain of PLC-gamma1 in mitogenic signaling. In this report, we provide evidence that SOS1, a p21Ras-specific guanine nucleotide exchange factor, directly binds to the SH3 domain of PLC-gamma1, and that the SH3 domain of PLC-gamma1 is involved in SOS1-mediated p21Ras activation. SOS1 was coprecipitated with the GST-fused SH3 domain of PLC-gamma1 in vitro. The interaction between SOS1 and the PLC-gamma1 SH3 domain is mediated by direct physical interaction. The carboxyl-terminal proline-rich domain of SOS1 is involved in the interaction with the PLC-gamma1 SH3 domain. Moreover, PLC-gamma1 could be co-immunoprecipitated with SOS1 antibody in cell lysates. From transient expression studies, we could demonstrate that the SH3 domain of PLC-gamma1 is necessary for the association with SOS1 in vivo. Intriguingly, overexpression of the SH3 domain of PLC-gamma1, lipase-inactive PLC-gamma1, or wild-type PLC-gamma1 elevated p21Ras activity and ERK activity when compared with vector transfected cells. The PLC-gamma1 mutant lacking the SH3 domain could not activate p21Ras. p21Ras activities in cell lines overexpressing either PLC-gamma1 or the SH2-SH2-SH3 domain of PLC-gamma1 were elevated about 2-fold compared to vector transfected cells. This study is the first to demonstrate that the PLC-gamma1 SH3 domain enhances p21Ras activity, and that the SH3 domain of PLC-gamma1 may be involved in the SOS1-mediated signaling pathway.