RESUMEN
CONTEXT: Mitochondrial capacity and metabolic potential are under the control of hormones, such as thyroid hormones. The most proximal regulator of the hypothalamic-pituitary-thyroid (HPT) axis, TRH, is the key hypothalamic integrator of energy metabolism via its impact on thyroid hormone secretion. OBJECTIVE: Here, we asked whether TRH directly modulates mitochondrial functions in normal, TRH-receptor-positive human epidermis. METHODS: Organ-cultured human skin was treated with TRH (5-100 ng/ml) for 12-48 h. RESULTS: TRH significantly increased epidermal immunoreactivity for the mitochondria-selective subunit I of respiratory chain complex IV (MTCO1). This resulted from an increased MTCO1 transcription and protein synthesis and a stimulation of mitochondrial biogenesis as demonstrated by transmission electron microscopy and TRH-enhanced mitochondrial DNA synthesis. TRH also significantly stimulated the transcription of several other mitochondrial key genes (TFAM, HSP60, and BMAL1), including the master regulator of mitochondrial biogenesis (PGC-1α). TRH significantly enhanced mitochondrial complex I and IV enzyme activity and enhanced the oxygen consumption of human skin samples, which shows that the stimulated mitochondria are fully vital because the main source for cellular oxygen consumption is mitochondrial endoxidation. CONCLUSIONS: These findings identify TRH as a potent, novel neuroendocrine stimulator of mitochondrial activity and biogenesis in human epidermal keratinocytes in situ. Thus, human epidermis offers an excellent model for dissecting neuroendocrine controls of human mitochondrial biology under physiologically relevant conditions and for exploring corresponding clinical applications.
Asunto(s)
Epidermis/metabolismo , Mitocondrias/metabolismo , Hormona Liberadora de Tirotropina/metabolismo , Adulto , Metabolismo Energético , Humanos , Persona de Mediana Edad , Técnicas de Cultivo de Órganos , Receptores de Hormona Liberadora de Tirotropina/metabolismoRESUMEN
In amphibians, thyrotropin-releasing hormone (TRH) stimulates skin melanophores by inducing secretion of α-melanocyte-stimulating hormone in the pituitary gland. However, it is unknown whether this tripeptide neurohormone exerts any direct effects on pigment cells, namely, on human melanocytes, under physiological conditions. Therefore, we have investigated whether TRH stimulates pigment production in organ-cultured human hair follicles (HFs), the epithelium of which expresses both TRH and its receptor, and/or in full-thickness human skin in situ. TRH stimulated melanin synthesis, tyrosinase transcription and activity, melanosome formation, melanocyte dendricity, gp100 immunoreactivity, and microphthalmia-associated transcription factor expression in human HFs in a pituitary gland-independent manner. TRH also stimulated proliferation, gp100 expression, tyrosinase activity, and dendricity of isolated human HF melanocytes. However, intraepidermal melanogenesis was unaffected. As TRH upregulated the intrafollicular production of "pituitary" neurohormones (proopiomelanocortin transcription and ACTH immunoreactivity) and as agouti-signaling protein counteracted TRH-induced HF pigmentation, these pigmentary TRH effects may be mediated in part by locally generated melanocortins and/or by MC-1 signaling. Our study introduces TRH as a novel, potent, selective, and evolutionarily highly conserved neuroendocrine factor controlling human pigmentation in situ. This physiologically relevant and melanocyte sub-population-specific neuroendocrine control of human pigmentation deserves clinical exploration, e.g., for preventing or reversing hair graying.
Asunto(s)
Color del Cabello/fisiología , Folículo Piloso/fisiología , Hormona Liberadora de Tirotropina/fisiología , Hormona Adrenocorticotrópica/metabolismo , Proteína de Señalización Agouti/metabolismo , Proliferación Celular/efectos de los fármacos , Femenino , Color del Cabello/efectos de los fármacos , Folículo Piloso/efectos de los fármacos , Humanos , Melaninas/biosíntesis , Melanocortinas/metabolismo , Melanocitos/metabolismo , Factor de Transcripción Asociado a Microftalmía/metabolismo , Monofenol Monooxigenasa/metabolismo , Técnicas de Cultivo de Órganos , Proopiomelanocortina/metabolismo , Receptores de Hormona Liberadora de Tirotropina/metabolismo , Hormona Liberadora de Tirotropina/farmacología , Antígeno gp100 del Melanoma/metabolismoRESUMEN
Several elements of the hypothalamic-pituitary-thyroid axis (HPT) reportedly are transcribed by human skin cell populations, and human hair follicles express functional receptors for TSH. Therefore, we asked whether the epidermis of normal human skin is yet another extrathyroidal target of TSH and whether epidermis even produces TSH. If so, we wanted to clarify whether intraepidermal TSH expression is regulated by TRH and/or thyroid hormones and whether TSH alters selected functions of normal human epidermis in situ. TSH and TSH receptor (TSH-R) expression were analyzed in the epidermis of normal human scalp skin by immunohistochemistry and PCR. In addition, full-thickness scalp skin was organ cultured and treated with TSH, TRH, or thyroid hormones, and the effect of TSH treatment on the expression of selected genes was measured by quantitative PCR and/or quantitative immunohistochemistry. Here we show that normal human epidermis expresses TSH at the mRNA and protein levels in situ and transcribes TSH-R. It also contains thyrostimulin transcripts. Intraepidermal TSH immunoreactivity is up-regulated by TRH and down-regulated by thyroid hormones. Although TSH-R immunoreactivity in situ could not be documented within the epidermis, but in the immediately adjacent dermis, TSH treatment of organ-cultured human skin strongly up-regulated epidermal expression of involucrin, loricrin, and keratins 5 and 14. Thus, normal human epidermis in situ is both an extrapituitary source and (possibly an indirect) target of TSH signaling, which regulates defined epidermal parameters. Intraepidermal TSH expression appears to be regulated by the classical endocrine controls that determine the systemic HPT axis.
Asunto(s)
Epidermis/metabolismo , Cuero Cabelludo/metabolismo , Hormona Liberadora de Tirotropina/metabolismo , Tirotropina/metabolismo , Tiroxina/metabolismo , Triyodotironina/metabolismo , Adulto , Anciano , Apoptosis/fisiología , Regulación hacia Abajo/fisiología , Epidermis/efectos de los fármacos , Femenino , Humanos , Sistema Hipotálamo-Hipofisario/metabolismo , Inmunohistoquímica , Queratina-14/genética , Queratina-14/metabolismo , Queratina-5/genética , Queratina-5/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Persona de Mediana Edad , Técnicas de Cultivo de Órganos , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Tirotropina/genética , Receptores de Tirotropina/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Cuero Cabelludo/efectos de los fármacos , Estadísticas no Paramétricas , Tirotropina/genética , Tirotropina/farmacología , Regulación hacia Arriba/fisiologíaRESUMEN
Here we demonstrate that the neuropeptide hormone thyrotropin (TSH), which controls thyroid hormone production, exerts a major nonclassical function in mitochondrial biology. Based on transcriptional, ultrastructural, immunohistochemical, and biochemical evidence, TSH up-regulates mitochondrial biogenesis and consequently activity in organ-cultured normal human epidermis in situ. Mitochondrial activity was assessed by measuring 2 key components of the respiratory chain. The abundance of mitochondria was assessed employing 2 independent morphological techniques: counting their numbers in human epidermis by high-magnification light microscopy of skin sections immunostained for mitochondria-selective cytochrome-c-oxidase subunit 1 (MTCO1) and transmission electron microscopy (TEM). Treatment with 10 mU/ml of TSH for 6 d strongly up-regulates the number of light-microscopically visualized, MTCO1-demarcated mitochondria. On the ultrastructural level, TEM confirms that TSH indeed stimulates mitochondrial proliferation and biogenesis in the perinuclear region of human skin epidermal keratinocytes. On the transcriptional level, TSH up-regulates MTCO1 mRNA (quantitative RT-PCR) and significantly enhances complex I and IV (cytochrome-c-oxidase) activity. This study pioneers the concept that mitochondrial energy metabolism and biogenesis in a normal, prototypic human epithelial tissue underlies potent neuroendocrine controls and introduces human skin organ culture as a clinically relevant tool for further exploring this novel research frontier in the control of mitochondrial biology.
Asunto(s)
Metabolismo Energético , Epidermis/metabolismo , Mitocondrias/metabolismo , Tirotropina/metabolismo , Células Cultivadas , Epidermis/efectos de los fármacos , Epidermis/ultraestructura , Expresión Génica/efectos de los fármacos , Humanos , Mitocondrias/efectos de los fármacos , Tirotropina/farmacologíaRESUMEN
Thyrotropin-releasing hormone (TRH) is the most proximal component of the hypothalamic-pituitary-thyroid axis that regulates thyroid hormone synthesis. Since transcripts for members of this axis were detected in cultured normal human skin cells and since human hair follicles (HFs) respond to stimulation with thyrotropin, we now have studied whether human HF functions are also modulated by TRH. Here we report that the epithelium of normal human scalp HFs expresses not only TRH receptors (TRH-R) but also TRH itself at the gene and protein level. Stimulation of microdissected, organ-cultured HFs with TRH promotes hair-shaft elongation, prolongs the hair cycle growth phase (anagen), and antagonizes its termination by TGF-beta2. It also increases proliferation and inhibits apoptosis of hair matrix keratinocytes. These TRH effects may be mediated in part by reducing the ATM/Atr-dependent phosphorylation of p53. By microarray analysis, several differentially up- or down-regulated TRH-target genes were detected (e.g., selected keratins). Thus, human scalp HFs are both a source and a target of TRH, which operates as a potent hair-growth stimulator. Human HFs provide an excellent discovery tool for identifying and dissecting nonclassical functions of TRH and TRH-mediated signaling in situ, which emerge as novel players in human epithelial biology.
Asunto(s)
Cabello/crecimiento & desarrollo , Hormona Liberadora de Tirotropina/fisiología , Apoptosis , Femenino , Regulación de la Expresión Génica , Folículo Piloso/efectos de los fármacos , Folículo Piloso/crecimiento & desarrollo , Humanos , Queratinocitos/citología , Persona de Mediana Edad , Técnicas de Cultivo de Órganos , Receptores de Tirotropina/efectos de los fármacos , Receptores de Tirotropina/metabolismoRESUMEN
The organ culture of human scalp hair follicles (HFs) is the best currently available assay for hair research in the human system. In order to determine the hair growth-modulatory effects of agents in this assay, one critical read-out parameter is the assessment of whether the test agent has prolonged anagen duration or induced catagen in vitro. However, objective criteria to distinguish between anagen VI HFs and early catagen in human HF organ culture, two hair cycle stages with a deceptively similar morphology, remain to be established. Here, we develop, document and test an objective classification system that allows to distinguish between anagen VI and early catagen in organ-cultured human HFs, using both qualitative and quantitative parameters that can be generated by light microscopy or immunofluorescence. Seven qualitative classification criteria are defined that are based on assessing the morphology of the hair matrix, the dermal papilla and the distribution of pigmentary markers (melanin, gp100). These are complemented by ten quantitative parameters. We have tested this classification system by employing the clinically used topical hair growth inhibitor, eflornithine, and show that eflornithine indeed produces the expected premature catagen induction, as identified by the novel classification criteria reported here. Therefore, this classification system offers a standardized, objective and reproducible new experimental method to reliably distinguish between human anagen VI and early catagen HFs in organ culture.
Asunto(s)
Folículo Piloso/anatomía & histología , Técnicas de Cultivo de Órganos/métodos , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Eflornitina/farmacología , Femenino , Cabello/efectos de los fármacos , Cabello/crecimiento & desarrollo , Folículo Piloso/efectos de los fármacos , Folículo Piloso/crecimiento & desarrollo , Folículo Piloso/metabolismo , Humanos , Antígeno Ki-67/metabolismoRESUMEN
Pituitary thyroid-stimulating hormone (TSH) regulates thyroid hormone synthesis via receptors (TSH-R) expressed on thyroid epithelial cells. As the hair follicle (HF) is uniquely hormone-sensitive and, hypothyroidism with its associated, increased TSH serum levels clinically can lead to hair loss, we asked whether human HFs are a direct target for TSH. Here, we report that normal human scalp skin and microdissected human HFs express TSH-R mRNA. TSH-R-like immunoreactivity is limited to the mesenchymal skin compartments in situ. TSH may alter HF mesenchymal functions, as it upregulates alpha-smooth muscle actin expression in HF fibroblasts. TSH-R stimulation by its natural ligand in organ culture changes the expression of several genes of human scalp HFs (for example keratin K5), upregulates the transcription of classical TSH target genes and enhances cAMP production. Although the functional role of TSH in human HF biology awaits further dissection, these findings document that intracutaneous TSH-Rs are fully functional in situ and that HFs of female individuals are direct targets for nonclassical, extrathyroidal TSH bioregulation. This suggests that organ-cultured scalp HFs provide an instructive and physiologically relevant human model for exploring nonclassical functions of TSH, in and beyond the skin.
Asunto(s)
Folículo Piloso/fisiología , Cuero Cabelludo/citología , Piel/citología , Tirotropina/fisiología , Actinas/metabolismo , Proliferación Celular/efectos de los fármacos , Células Cultivadas , AMP Cíclico/metabolismo , Femenino , Fibroblastos/metabolismo , Folículo Piloso/efectos de los fármacos , Folículo Piloso/crecimiento & desarrollo , Humanos , Queratina-5/metabolismo , Mesodermo/citología , Mesodermo/metabolismo , Proteínas Nucleares/metabolismo , Técnicas de Cultivo de Órganos , ARN Mensajero/metabolismo , Receptores de Tirotropina/metabolismo , Cuero Cabelludo/metabolismo , Piel/metabolismo , Tiroglobulina/metabolismo , Factor Nuclear Tiroideo 1 , Tirotropina/farmacología , Factores de Transcripción/metabolismoRESUMEN
CONTEXT: Both insufficient and excess levels of thyroid hormones (T3 and T4) can result in altered hair/skin structure and function (e.g. effluvium). However, it is still unclear whether T3 and T4 exert any direct effects on human hair follicles (HFs), and if so, how exactly human HFs respond to T3/T4 stimulation. OBJECTIVE: Our objective was to asses the impact of T3/T4 on human HF in vitro. METHODS: Human anagen HFs were isolated from skin obtained from females undergoing facelift surgery. HFs from euthyroid females between 40 and 69 yr (average, 56 yr) were cultured and treated with T3/T4. RESULTS: Studying microdissected, organ-cultured normal human scalp HFs, we show here that T4 up-regulates the proliferation of hair matrix keratinocytes, whereas their apoptosis is down-regulated by T3 and T4. T4 also prolongs the duration of the hair growth phase (anagen) in vitro, possibly due to the down-regulation of TGF-beta2, the key anagen-inhibitory growth factor. Because we show here that human HFs transcribe deiodinase genes (D2 and D3), they may be capable of converting T4 to T3. Intrafollicular immunoreactivity for the recognized thyroid hormone-responsive keratins cytokeratin (CK) 6 and CK14 is significantly modulated by T3 and T4 (CK6 is enhanced, CK14 down-regulated). Both T3 and T4 also significantly stimulate intrafollicular melanin synthesis. CONCLUSIONS: Thus, we present the first evidence that human HFs are direct targets of thyroid hormones and demonstrate that T3 and/or T4 modulate multiple hair biology parameters, ranging from HF cycling to pigmentation.
Asunto(s)
Color del Cabello/efectos de los fármacos , Folículo Piloso/fisiología , Queratinocitos/citología , Tiroxina/farmacología , Triyodotironina/farmacología , Adulto , Anciano , División Celular/efectos de los fármacos , Femenino , Folículo Piloso/citología , Folículo Piloso/efectos de los fármacos , Humanos , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Queratinocitos/efectos de los fármacos , Antígeno Ki-67/metabolismo , Persona de Mediana Edad , Técnicas de Cultivo de Órganos , Factor de Crecimiento Transformador beta2/metabolismoRESUMEN
PURPOSE: In radioiodine therapy the "stunning phenomenon" is defined as a reduction of radioiodine uptake after diagnostic application of (131)I. In the current study, we established an in vitro model based on the "Fisher rat thyrocyte cell line no. 5" (FRTL-5) to investigate the stunning. METHODS: TSH-stimulated FRTL-5 cells were incubated with (131)I. Time-dependent (131)I uptake and the viability of FRTL-5 cells were evaluated at 4-144 h after radioiodine application. All data was corrected for number of viable cells, half life and (131)I concentration. Sodium iodide symporter (NIS) and the housekeeping gene (beta-actin, GAPDH) levels were quantified by quantitative polymerase chain reaction (qPCR). Additionally, immunohistochemical staining (IHC) of NIS on the cell membrane was carried out. RESULTS: FRTL-5 monolayer cell cultures showed a specific maximum uptake of (131)I 24-48 h after application. Significantly decreased (131)I uptake values were observed after 72-144 h. The decrease in radioiodine uptake was correlated with decreasing mRNA levels of NIS and housekeeping genes. In parallel, unlike in controls, IHC staining of NIS on FRTL-5 cells declined significantly after (131)I long-term incubation. CONCLUSIONS: It could be demonstrated that during (131)I incubation of FRTL-5 cells, radioiodine uptake decreased significantly. Simultaneously decreasing levels of NIS mRNA and protein expression suggest a NIS-associated mechanism. Since mRNA levels of housekeeping genes decreased, too, the reduced NIS expression might be provoked by a cell cycle arrest. Our investigations recommend the FRTL-5 model as a valuable tool for further molecular biological investigations of the stunning phenomenon.