RNF167 targets Arl8B for degradation to regulate lysosome positioning and endocytic trafficking.

The protease-associated (PA) domain-containing E3 ubiquitin ligases are transmembrane proteins located in intracellular organelles such as the endoplasmic reticulum, endosomes, or lysosomes. The functional roles of these ubiquitin ligases are not well defined. To understand the function of E3 ubiquitin ligases, identification of their substrates is of critical importance. In this study, we describe a newly devised method based on proximity-dependent biotin labeling to identify substrates of ubiquitin ligases. Application of this method to RING finger protein 167 (RNF167), a member of the PA domain-containing E3 family, led to identification of Arl8B as its substrate. We demonstrated that RNF167 ubiquitinates Arl8B at the lysine residue K141 and reduces the level of the Arl8B protein. Overexpression and knockdown of RNF167 revealed its regulatory role in Arl8B-dependent lysosome positioning and endocytic trafficking to lysosomes. Furthermore, we found that the ubiquitination-defective Arl8B K141R mutant counteracts RNF167 in these cellular events. These results indicate that RNF167 plays a crucial role as an E3 ubiquitin ligase targeting Arl8B to regulate lysosome positioning and endocytic trafficking.

Increased expression of Dkk1 by HR is associated with alteration of hair cycle in hairpoor mice.

BACKGROUND: Hairless (Hr), a transcriptional corepressor expressed mainly in the skin, regulates hair follicle (HF) morphogenesis and hair cycling. Recently, we reported a new Hr mutant mouse, "Hairpoor" (Hr(Hp)), that resembles the human hair disorder Marie Unna hereditary hypotrichosis (MUHH) in the heterozygous state. The Wnt/ß-catenin signaling pathway is critical for homeostasis in various adult tissues including skin and HFs. One of the Wnt inhibitors, Dickkopf (Dkk), inhibits hair growth during the hair cycle as a catagen inducer of apoptosis, resulting in HF reductions. OBJECTIVE: To investigate regulation of Dkk1 by HR and its effect on hair formation. METHODS: The relative expression of Dkk1 in (+)/Hr(HP) and Hr(Hp)/Hr(Hp) mice during the hair cycle was investigated using real time PCR and Western blot analysis. Immunohistochemistry was performed in order to confirm abnormal expression of Dkk1 in HFs of (+)/Hr(HP) and Hr(Hp)/Hr(Hp) mice. To determine whether Dkk1 expression was also regulated by HR in vitro, an Hr-transient transfection experiment was performed. Alteration of the hair cycle in Hr(HP) heterozygous mice was identified by determination of the hair cycle and measurement of HF length. RESULTS: Dkk1 expression was increased in the skin of (+)/Hr(HP) and Hr(Hp)/Hr(Hp) mice, as well as in Hr-overexpressing mouse keratinocytes. Additionally, an earlier entrance of HFs into catagen and shortened HF length in (+)/Hr(HP) mice compared to wild-type mice was observed. CONCLUSION: Study results suggested that up-regulation of Dkk1 by HR contributed to abnormal development of HFs and failure in regeneration of HFs in Hr(Hp)/Hr(Hp) mice. These findings also indicated that alteration of the hair cycle in (+)/Hr(HP) mice was related to the up-regulation of Dkk1 by HR.

Hairless down-regulates expression of Msx2 and its related target genes in hair follicles.

BACKGROUND: Hairless (HR), a transcriptional cofactor, plays important roles in hair follicle (HF) morphogenesis and cycling. Recently, we reported the new Hr mutant mouse called "Hairpoor" (Hr(Hp)) that causes HR overexpression through translational de-repression. The Msh homeobox 2 (Msx2) is a homolog of the Drosophila muscle segment homeobox (msh) gene, which expressed in the hair bulb, including in the germinal matrix, and its expression spreads into the upper region of the HF including the hair cortex. OBJECTIVE: Although Msx2 is regarded as an important gene in hair cycle control and hair shaft differentiation, the regulation of Msx2 expression is not well-known. METHODS: Using realtime polymerase chain reaction (PCR) and western blot, we investigated the relationship between HR and Msx2 in the Hr(Hp)/Hr(Hp) mouse during the HF morphogenesis. Immunohistochemistry was performed to compare the pattern of expression of MSX2 in Hr(Hp)/Hr(Hp) mouse skin with that in wild-type skin. Msx2 mRNA expression and promoter activity was estimated using a transient expression system to see whether HR down-regulates Msx2 expression in vitro. We also investigated whether downregulation of MSX2 by HR also affects the MSX2 regulatory pathway in the Hr(Hp)/Hr(Hp) mouse and in an in vitro system. RESULTS: We found that the expression of Msx2 was down-regulated by HR, which in turn down-regulated expression of Foxn1 and Lef1, MSX2 target genes, in vivo as well as in vitro. CONCLUSION: Our results show that HR regulates expression of genes in the MSX2 regulatory pathway, which explains abnormal HF formation in Hr(Hp)/Hr(Hp) skin.

Gene expression profile of the skin in the 'hairpoor' (HrHp) mice by microarray analysis.

BACKGROUND: The transcriptional cofactor, Hairless (HR), acts as one of the key regulators of hair follicle cycling; the loss of function mutations is the cause of the expression of the hairless phenotype in humans and mice. Recently, we reported a new Hr mutant mouse called 'Hairpoor' (Hr(Hp)). These mutants harbor a gain of the function mutation, T403A, in the Hr gene. This confers the overexpression of HR and Hr(Hp) is an animal model of Marie Unna hereditary hypotrichosis in humans. In the present study, the expression profile of Hr(Hp)/Hr(Hp) skin was investigated using microarray analysis to identify genes whose expression was affected by the overexpression of HR. RESULTS: From 45,282 mouse probes, differential expressions in 43 (>2-fold), 306 (>1.5-fold), and 1861 genes (>1.2-fold) in skin from Hr(Hp)/Hr(Hp) mice were discovered and compared with skin from wild-type mice. Among the 1861 genes with a > 1.2-fold increase in expression, further analysis showed that the expression of eight genes known to have a close relationship with hair follicle development, ascertained by conducting real-time PCR on skin RNA produced during hair follicle morphogenesis (P0-P14), indicated that four genes, Wif1, Casp14, Krt71, and Sfrp1, showed a consistent expression pattern with respect to HR overexpression in vivo. CONCLUSION: Wif1 and Casp14 were found to be upregulated, whereas Krt71 and Sfrp1 were downregulated in cells overexpressing HR in transient transfection experiments on keratinocytes, suggesting that HR may transcriptionally regulate these genes. Further studies are required to understand the mechanism of this regulation by the HR cofactor.