Pioneering studies on monogenic central precocious puberty

ABSTRACT Pubertal timing in humans is determined by complex interactions including hormonal, metabolic, environmental, ethnic, and genetic factors. Central precocious puberty (CPP) is defined as the premature reactivation of the hypothalamic-pituitary-gonadal axis, starting before the ages of 8 and 9 years in girls and boys, respectively; familial CPP is defined by the occurrence of CPP in two or more family members. Pioneering studies have evidenced the participation of genetic factors in pubertal timing, mainly identifying genetic causes of CPP in sporadic and familial cases. In this context, rare activating mutations were identified in genes of the kisspeptin excitatory pathway (KISS1R and KISS1 mutations). More recently, loss-of-function mutations in two imprinted genes (MKRN3 and DLK1) have been identified as important causes of familial CPP, describing novel players in the modulation of the hypothalamic-pituitary-gonadal axis in physiological and pathological conditions. MKRN3 mutations are the most common cause of familial CPP, and patients with MKRN3 mutations present clinical features indistinguishable from idiopathic CPP. Meanwhile, adult patients with DLK1 mutations present high frequency of metabolic alterations (overweight/obesity, early onset type 2 diabetes and hyperlipidemia), indicating that DLK1 may be a novel link between reproduction and metabolism. Arch Endocrinol Metab. 2019;63(4):438-44

Effect of Dlk1 and Jagged1 gene on the proliferation and transdifferentiation of the primary type Ⅱ alveolar epithelial cells / 中华实用儿科临床杂志

Objective To study the effects of the Notch ligands Dlk1 and recombinant human nucleu factorκB (Jagged1) on the proliferation and transdifferentiation of the type Ⅱ alveolar epithelial cells when the Notch signaling pathway activated.Methods The primary type Ⅱ alveolar epithelial cells (AEC Ⅱ) cultured with recombinant protein Dlk1 and recombinant human nucleu factor-κB (rhNF-κB) (activator of Jagged1),respectively,and then cultured with DMEM (containing 120 mL/L FBS) as controls.Proliferation and differentiation conditions of the AEC Ⅱ were observed at 48 h,72 h,96 h time point by the light microscope and electron microscopes separately.Cell number was counted with hemacytometer; the proliferation rate was measured by methyl thiazolyl tetrazolium (MTT) ; Immunofluorescence double standard method was used to detect the AEC Ⅱ specific surfactant protein C (SP-C) and AEC Ⅰ specific protein aquaporin5 (AQPS) ;the expression of SP-C,AQPS,Dlk1,Jagged1,Notch1 and Hes1 mRNA were detected by real time-PCR.Results The cell population and proliferation:compared with control group,AEC Ⅱ proliferation was promoted in the Dlk1 group [cell numbers (× 109/L) 9.05 ± 0.45 vs 7.95 ± 0.65,11.68 ± 0.43 vs 8.68 ± 0.52,11.55 ± 0.17 vs 8.73 ± 0.48,all P ＜ 0.05 ; MTT results (value A) 0.699 ± 0.050 vs 0.462 ± 0.080,0.912 ± 0.080 vs 0.535 ±0.040,0.726 ±0.050 vs 0.540 ±0.020,all P ＜0.05] and decelerated AEC Ⅱ transdifferentiation into AEC Ⅰ ; while AEC Ⅱ proliferation was inhibited in rhNF-κB group [cell numbers (× 109/L) 4.95 ± 0.33 vs 7.95 ± 0.65,4.73 ±0.71 vs 8.68 ± 0.52,4.04 ± 0.11 vs 8.73 ± 0.48,all P ＜ 0.05; MTT results (value A) 0.398 ± 0.030 vs 0.462 ± 0.080,0.402 ± 0.070 vs 0.535 ± 0.040,0.380 ± 0.110 vs 0.540 ± 0.020,all P ＜ 0.05] and accelerated AEC Ⅱ transdifferentiation into AEC Ⅰ.One-Way ANOVA showed that the difference among the 3 groups had statistical significance (cell numbers:F =486.73,P =0.02; cell proliferation:F =37.16,P =0.02).The mRNA expression:compared with control group,the expression of SP-C mRNA of Dlk1 group was significantly higher (P ＜ 0.05) while the expression of AQP5 mRNA was remarkably lower and delayed (P ＜ 0.05),the expression of Jagged1 mRNA was weak or little,Dlk1 and Notch1 mRNA were up-regulated (P ＜ 0.05),and the Hes1 mRNA was reduced (P ＜ 0.05) ; the expression of SP-C mRNA of rhNF-κB group was significantly reduced (P ＜ 0.05),while the AQP5 mRNA expressed ahead of time and increased (P ＜ 0.05),Jagged1,Hes1 and Notch1 mRNA were higher (P ＜ 0.05),and the Dlk1 mRNA was weak.One-Way ANOVA showed that the difference in the expressions of SP-C,AQP5,D1k1,Jagged1,Hes1 and Notch1 mRNA among the 3 groups had staistical significance (F =96.80,P =0.01 ; F =82.55,P =0.01 ; F =269.80,P=0.00;F =312.34,P =0.00;F =169.17,P =0.01;F =19.85,P =0.02).Conclusions There are varied effects on proliferation and differentiation of the AEC Ⅱ when the Notch signaling is activated by different ligands:Dlk1 promoted proliferation and inhibited differentiation,while Jagged1 inhibited proliferation and promoted transdifferentiation.

TET2 and DLK1 gene expression and their clinical significance in bone marrow CD3+ T cells of patients with myelodysplastic syndrome / 中华内科杂志

Objective To investigate the expression of TET2 and DLK1 mRNA in bone marrow CD3+ T cells of patients with myelodysplastic syndrome (MDS) and their clinical significance and to explore the potential mechanism of abnormal cell-mediated immunity.Methods CD3+ T cells were sorted by magnetic activated cell-sorting system.The expressions of TET2 and DLK1 mRNA in bone marrow CD3+ T cells from 26 MDS patients and 16 healthy controls were detected by fluorescence quantitative PCR.Results The expression of TET2 mRNA in CD3+ T cells was down-regulated in the MDS patients by (0.16 ±0.15) fold compared with the controls ( P ＜ 0.05 ).The expression of TET2 mRNA in CD3+ T cells of MDS patients was positively correlated with serum complement C3 ( r =0.404,P ＜ 0.05 ).The expression of DLK1 mRNA in CD3+ T cells was up-regulated in the MDS patients by (1.61 ±0.88) folds compared with the controls (P＜0.05).Grouped by the chromosomes,the patients with chromosome abnormalities presented significantly higher DLK1 mRNA level than those with normal chromosomes [ ( 1.45 ± 0.44 ) folds,P ＜0.05 ].The expression of DLK1 mRNA in CD3+ T cells of MDS patients was positively correlated with the proportion of bone marrow blasts ( r =0.343,P ＜ 0.05 ).Conclusions The mRNA expression of TET2 in CD3+ T cells of MDS patients was decreased while the mRNA expression of DLK1 was increased,which might decline the immune surveillance function.The findings would be useful for exploring the mechanism of immune tolerance.

Expression of DLK1 protein and its correlation with renal cell carcinoma pathological characteristics / 中华泌尿外科杂志

Objective To identify the expression of DLK1 protein in different types of renal cell carcinomas and its correlations with pathological characteristics and metastasis. Methods Immunohistochemistry analysis was performed to evaluate the expression of DLK1 protein in 94 cases of primary clear cell renal cell carcinoma, 76 cases of papillary renal cell carcinoma, 45 cases of chromophobe renal cell carcinoma, 71 cases of distal metastatic and 24 cases of lymph node metastatic clear cell renal cell carcinoma, as well as 18 cases of normal renal tissue. The correlations of DLK1 protein expression with pathological characteristics were analyzed. Results DLK1 protein was expressed in proximal and distal renal tubular epithelial cells in all the normal renal cases. In contrast, DLK1 protein expression was lower in different types of renal cell carcinoma. The low or negative expression of DLK1 protein in clear cell renal cell carcinoma, papillary renal cell carcinoma and chromophobe renal cell carcinoma was 33.0% (31/94), 27.6% (21/76) and 33.3% (15/45), respectively. Compared to normal renal tissue, DLK1 protein expression was significantly down-regulated in renal cell carcinomas (P＞0.05), whereas there was no significant difference on DLK1 protein expressions among the different types (P＞0.05) of renal cell carcinomas. DLK1 protein expression was not correlated with sex (60 male and 34 female cases), age (≥55, 50 cases and 55, 44 cases), grade (41 cases in grade I, 9 cases in grade II, 21 cases in grade III and 23 cases in grade Ⅳ respectively) and lymph node metastasis (76 cases with and 18 cases without lymph node metastasis) in clear cell renal cell carcinoma (P＞0.05). There was also no significant difference among primary, lymph node and distal metastatic lesions of clear cell carcinoma (P＞0.05). Conclusions DLK1 protein expression is commonly down-regulated in different types of renal cell carcinomas. Down-regulation of DLK1 protein expression is not associated with pathological characteristics and metastasis in clear cell renal cell carcinoma.

Expression of DLK1 and MEG3 genes in porcine tissues during postnatal development

The Drosophila-like homolog 1 (DLK1), a transmembrane signal protein similar to other members of the Notch/Delta/Serrate family, regulates the differentiation process in many types of mammalian cells. Callipyge sheep and DLK1 knockout mice are excellent examples of a fundamental role of the gene encoding DLK1 in muscle growth and fat deposition. DLK1 is located within co-regulated imprinted clusters (the DLK1/DIO3 domain), along with other imprinted genes. Some of these, e.g. the RNA coding MEG3 gene, presumedly interfere with DLK1 transcription. The aim of our study was to analyze DLK1 and MEG3 gene expression in porcine tissues (muscle, liver, kidney, heart, brain stem) during postnatal development. The highest expression of both DLK1 and MEG3 variant 1 (MEG3 var.1) was observed in the brain-stem and muscles, whereas that of MEG3 variant 2 (MEG3 var.2) was the most abundant in muscles and the heart. During development (between 60 and 210 days of age) expression of analyzed genes was down-regulated in all the tissues. An exception was the brain-stem, where there was no significant change in MEG3 (both variants) mRNA level, and relatively little decline (2-fold) in that of DLK1 transcription. This may indicate a distinct function of the DLK1 gene in the brain-stem, when compared with other tissues.

The effects of nutrient depleted microenvironments and delta-like 1 homologue (DLK1) on apoptosis in neuroblastoma

The tumor microenvironment, particularly sufficient nutrition and oxygen supply, is important for tumor cell survival. Nutrition deprivation causes cancer cell death. Since apoptosis is a major mechanism of neuronal loss, we explored neuronal apoptosis in various microenvironment conditions employing neuroblastoma (NB) cells. To investigate the effects of tumor malignancy and differentiation on apoptosis, the cells were exposed to poor microenvironments characterized as serum-free, low-glucose, and hypoxia. Incubation of the cells in serum-free and low-glucose environments significantly increased apoptosis in less malignant and more differentiated N-type IMR32 cells, whereas more malignant and less differentiated I-type BE(2)C cells were not affected by those treatments. In contrast, hypoxia (1% O2) did not affect apoptosis despite cell malignancy. It is suggested that DLK1 constitutes an important stem cell pathway for regulating self-renewal, clonogenicity, and tumorigenicity. This raises questions about the role of DLK1 in the cellular resistance of cancer cells under poor microenvironments, which cancer cells normally encounter. In the present study, DLK1 overexpression resulted in marked protection from apoptosis induced by nutrient deprivation. This in vitro model demonstrated that increasing severity of nutrition deprivation and knock-down of DLK1 caused greater apoptotic death, which could be a useful strategy for targeted therapies in fighting NB as well as for evaluating how nutrient deprived cells respond to therapeutic manipulation.

Effect of retinoic acid and delta-like 1 homologue (DLK1) on differentiation in neuroblastoma

The principal objective of this study was to evaluate the chemopreventive and therapeutic effects of a combination of all-trans-retinoic acid (RA) and knockdown of delta-like 1 homologue (Drosophila) (DLK1) on neuroblastoma, the most common malignant disease in children. As unfavorable neuroblastoma is poorly differentiated, neuroblastoma cell was induced differentiation by RA or DLK1 knockdown. Neuroblastoma cells showed elongated neurite growth, a hallmark of neuronal differentiation at various doses of RA, as well as by DLK1 knockdown. In order to determine whether or not a combination of RA and DLK1 knockdown exerts a greater chemotherapeutic effect on neuroblastoma, cells were incubated at 10 nM RA after being transfected with SiRNA-DLK1. Neuronal differentiation was increased more by a combination of RA and DLK1 knockdown than by single treatment. Additionally, in order to assess the signal pathway of neuroblastoma differentiation induced by RA and DLK1 knockdown, treatment with the specific MEK/ERK inhibitors, U0126 and PD 98059, was applied to differentiated neuroblastoma cells. Differentiation induced by RA and DLK1 knockdown increased ERK phosphorylation. The MEK/ERK inhibitor U0126 completely inhibited neuronal differentiation induced by both RA and DLK1 knockdown, whereas PD98059 partially blocked neuronal differentiation. After the withdrawal of inhibitors, cellular differentiation was fully recovered. This study is, to the best of our knowledge, the first to demonstrate that the specific inhibitors of the MEK/ERK pathway, U0126 and PD98059, exert differential effects on the ERK phosphorylation induced by RA or DLK1 knockdown. Based on the observations of this study, it can be concluded that a combination of RA and DLK1 knockdown increases neuronal differentiation for the control of the malignant growth of human neuroblastomas, and also that both MEK1 and MEK2 are required for the differentiation induced by RA and DLK1 knockdown.