The clock gene brain and muscle Arnt-like protein-1 (BMAL1) is involved in hair growth.

It is known that baldness caused by androgenetic alopecia is involved with androgen and the androgen receptor. Furthermore, it has been reported that testosterone secretion follows a circadian rhythm. Therefore, we hypothesized that a relationship exists between androgen-induced alopecia and biological rhythm. The mammalian circadian rhythm is controlled by several clock genes. Brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein-1 (BMAL1), one of the clock genes, is a transcription factor that plays central roles in the regulation of circadian rhythms. In this study, we investigated the influence of BMAL1 on hair follicle functions and hair growth. Mice deficient in BMAL1 expression exhibited a delay in hair regrowth after shaving. In hair follicles of mouse vibrissa, expression of Bmal1 and other clock genes was found to be rhythmic. Knockdown of BMAL1 in human follicle dermal papilla cells resulted in modulation of expression of several hair growth-related genes. Therefore, we concluded that expression of clock genes in hair follicles is linked to the circadian rhythm and that BMAL1 can regulate hair growth.

Specific enhancement of vascular endothelial growth factor (VEGF) production in ischemic region by alprostadil--potential therapeutic application in pharmaceutical regenerative medicine.

Alprostadil (lipo-PGE1) is a drug delivery system preparation. This preparation is applied to treat refractory skin ulcers and arteriosclerosis obliterans. We investigated the effects of alprostadil by using the earflap ischemic model. The following results were obtained: 1) Treatment with alprostadil significantly increased the VEGF contents in an ischemic ear; 2) Treatment with alprostadil resulted in strongly expressed VEGF levels only in the ischemic region; 3) Image analysis revealed a significant increase in the number of vessel bypasses and paths after flap creation with alprostadil administration compared to the vehicle-treated ears. The results suggest that it may be possible to apply alprostadil as one device for regenerative medical technology.

Improvement of hind-limb paralysis following traumatic spinal cord injury in rats by grafting normal human keratinocytes: new cell-therapy strategy for nerve regeneration.

Somatic (adult) stem cells are thought to have pluripotency, just as do embryotic stem (ES) cells. We investigated the possibility that grafted epithelial keratinocytes could induce spinal cord regeneration in an animal model of spinal cord injury (SCI). Normal human keratinocytes were cultured by the routine technique, and normal human dermal fibroblasts were cultured by a similar method as a control group. SCI model was prepared by dropping a 10-g weight onto the exposed spinal cord of rats from a height of 25 mm, and 8 days later, the cultured cells were grafted into the injury site. Motor function was significantly improved in the cultured-keratinocyte-grafted group compared with that in the fibroblast-grafted group. After functional observation, human nestin- and nuclei-positive cells were found at the grafted spinal cord. Grafted cultured keratinocytes induced in vitro morphological changes in the neural induction medium. These results indicated one possibility that some of the grafted cultured keratinocytes survived and could have contributed to neural regeneration. On the other hand, it should be noted that the grafted cultured keratinocytes secreted a large amount of enzymes and/or growth factors. Therefore, another possibility is that the grafted-keratinocyte-derived factors could induce survived cell growth and endogenous neural differentiation of spinal-nerve-derived stem cells surrounding the injured spinal cord, leading to functional recovery. Epithelial stem cell therapy may be applied clinically in the near future to treat SCI.

Aryl hydrocarbon receptor functions as a potent coactivator of E2F1-dependent trascription activity.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates a spectrum of toxic and biological effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin and related compounds. Several reports have shown that the AhR plays an important role in the control of cell-cycle progression, and this function is thought to be partly associated with the tumor promotion activity of dioxin. However, the underling mechanisms are not fully understood. We have previously shown that overexpression of AhR, as well as AhR ligand treatment, stimulates cell proliferation of human lung cancer A549 cells. In AhR-activated cells, the expression levels of DNA synthesis-related genes such as proliferating cell nuclear antigen (PCNA) and RFC38 are notably increased. Expression of these genes is mainly regulated by E2F1, a transcription factor that is crucial for transition of the cell cycle from G1 to S phase. We show here that the transcriptional activity of E2F1 is increased by the AhR agonist treatment and that this effect depends on the presence of AhR. Functional mapping of AhR showed that the Per-Arnt-Sim (PAS) B domain is required for promotion of E2F1 activity. The mechanism involves formation of a complex of AhR and E2F1 on the regulatory region in the E2F1 target gene, followed by recruitment of coactivator activator for thyroid hormone and retinoid receptors (ACTR). Consequently, the results in this study indicate the physiological function of AhR as a potent transcriptional coactivator of E2F1-dependent transcription and implicate the AhR-E2F1 interaction as a part of the mechanism by which AhR/Arnt promotes cell proliferation.

Crosstalk between the AHR signaling pathway and circadian rhythm.

In this chapter, we review the crosstalk between the AHR signaling pathway and molecular clock system in mammals. In mammals, circadian rhythm is observed in most physiological functions including behavior, metabolism, cell growth, and immune responses. Circadian rhythm is regulated by a transcriptional feedback loop, and the transcription factor called "Brain Muscle ARNT-like protein 1 (BMAL1)" is a master regulator of this system. Because of its structural similarity to ARNT, a partner of AHR, BMAL1 is also referred as ARNT3. This structural feature of BMAL1 suggests that the activation of the AHR signaling pathway may influence the regulation of circadian rhythm. Several studies have shown that the expression levels of AHR display diurnal variation in many tissues. This circadian variation of AHR means that the pharmacological effects of AHR agonists vary according to the time of administration. AHR agonist administration results in a disruption of circadian rhythm with regard to behavior, immune cell proliferation, etc. As such, understanding the crosstalk between the AHR signaling and circadian rhythm may provide a new insight into TCDD actions.

Brain and muscle Arnt-like protein-1 (BMAL1), a component of the molecular clock, regulates adipogenesis.

Brain and muscle Arnt-like protein-1 (BMAL1; also known as MOP3 or Arnt3) is a transcription factor known to regulate circadian rhythm. Here, we established its involvement in the control of adipogenesis and lipid metabolism activity in mature adipocytes. During adipose differentiation in 3T3-L1 cells, the level of BMAL1 mRNA began to increase 4 days after induction and was highly expressed in differentiated cells. In white adipose tissues isolated from C57BL/6J mice, BMAL1 was predominantly expressed in a fraction containing adipocytes, as compared with the stromal-vascular fraction. BMAL1 knockout mice embryonic fibroblast cells failed to be differentiated into adipocytes. Importantly, adding BMAL1 back by adenovirus gene transfer restored the ability of BMAL1 knockout mice embryonic fibroblast cells to differentiate. Knock-down of BMAL1 expression in 3T3-L1 cells by an RNA interference technique allowed the cells to accumulate only minimum amounts of lipid droplets in the cells. Adenovirus-mediated expression of BMAL1 in 3T3-L1 adipocytes resulted in induction of several factors involved in lipogenesis. The promoter activity of these genes was stimulated in a BMAL1-dependent manner. Interestingly, expression of these factors showed clear circadian rhythm in mice adipose tissue. Furthermore, overexpression of BMAL1 in adipocytes increased lipid synthesis activity. These results indicate that BMAL1, a master regulator of circadian rhythm, also plays important roles in the regulation of adipose differentiation and lipogenesis in mature adipocytes.