Longitudinal skeletal growth and growth plate morphological characteristics of chondro-tissue specific CUL7 knockout mice.

BACKGROUND: 3 M syndrome is first reported in 1975，which characterized by severe pre- and postnatal growth retardation, skeletal malformation and facial dysmorphism. These three genes (CUL7, OBSL1 and CCDC8) have been identified to be respond for 3 M syndrome, of which CUL7 is accounting for approximately 70%. To date, the molecular mechanism underlying the pathogenesis of 3 M syndrome remains poorly understood. Previous studies showed that no Cul7-/- mice could survive after birth, because of growth retardation at late gestational stage and respiratory distress after birth. The establishment of the animal model of cartilage specific Cul7 knockout mice (Cul7fl/fl;Col2a1-CreERT2 mice) has confirmed that Cul7fl/fl;Col2a1-CreERT2 mice can be selective in a time- and tissue-dependent manner, which can provide an experimental basis for further research on severe genetic diseases related to growth plates. OBJECTIVE: To establish a model of Cul7fl/fl;Col2a1-CreERT2 mice based on Cre/LoxP system, and to further observe its phenotype and morphological changes in growth plate. METHODS: The Cul7fl/fl;Col2a1-CreERT2 mice were taken as the experimental group, while the genotype of Cul7fl/+;Col2a1-CreERT2 mice were used as the control group. The gross morphological features and X-ray films of limbs in the two groups were observed every week for 3-6 consecutive weeks, and the length of the mice from nose to the tail, the length of femur and tibia were recorded. In the meantime, The histological morphology of tibial growth plates was compared between the two groups. RESULTS: A preliminary model of Cul7fl/fl;Col2a1-CreERT2 mice was established. The Cul7fl/fl;Col2a1-CreERT2 mice had abnormally short and deformed limbs (P<0.05), increased thickness of growth plate, the disorderly arranged chondrocyte columns, decreased number of cells in the proliferation zone, changes in the shape from flat to round, obviously expanded extracellular matrix, and disordered arrangement, thickening and loosening of bone trabecula at the proximal metaphysis of the femur. CONCLUSIONS: The knockout of Cul7 gene may affect both the proliferation of chondrocytes and the endochondral osteogenesis, confirming that Cul7 is essential for the normal development of bone in the body.

Inhibition of LAMP3 mediates the protective effect of vitamin D against hypoxia/reoxygenation in trophoblast cells.

Inadequate invasion and excessive apoptosis of trophoblast cells are associated with the development of preeclampsia. Vitamin D deficiency in pregnant women may lead to an increased risk of preeclampsia. However, the underlying mechanisms by which vitamin D is effective in preventing preeclampsia are not fully understood. The objectives of this study were to investigate the role of lysosome-associated membrane glycoprotein 3 (LAMP3) in the pathogenesis of preeclampsia and to evaluate whether vitamin D supplementation would protect against the development of preeclampsia by regulating LAMP3 expression. Firstly, the mRNA and protein levels of LAMP3 were significantly upregulated in the placentas of preeclampsia patients compared to normal placentas, especially in trophoblast cells (a key component of the human placenta). In the hypoxia/reoxygenation (H/R)-exposed HTR-8/Svneo trophoblast cells, LAMP3 expression was also upregulated. H/R exposure repressed cell viability and invasion and increased apoptosis of trophoblast cells. siRNA-mediated knockdown of LAMP3 increased cell viability and invasion and suppressed apoptosis of H/R-exposed trophoblast cells. We further found that 1,25(OH)2D3 (the hormonally active form of vitamin D) treatment reduced LAMP3 expression in H/R exposed trophoblast cells. In addition, 1,25(OH)2D3 treatment promoted cell viability and invasion and inhibited apoptosis of H/R-exposed trophoblast cells. Notably, overexpression of LAMP3 abrogated the protective effect of 1,25(OH)2D3 on H/R-exposed trophoblast cells. Collectively, we demonstrated trophoblast cytoprotection by vitamin D, a process mediated via LAMP3.

Inhibition of LAMP3 mediates the protective effect of vitamin D against hypoxia/reoxygenation in trophoblast cells

Inadequate invasion and excessive apoptosis of trophoblast cells are associated with the development of preeclampsia. Vitamin D deficiency in pregnant women may lead to an increased risk of preeclampsia. However, the underlying mechanisms by which vitamin D is effective in preventing preeclampsia are not fully understood. The objectives of this study were to investigate the role of lysosome-associated membrane glycoprotein 3 (LAMP3) in the pathogenesis of preeclampsia and to evaluate whether vitamin D supplementation would protect against the development of preeclampsia by regulating LAMP3 expression. Firstly, the mRNA and protein levels of LAMP3 were significantly upregulated in the placentas of preeclampsia patients compared to normal placentas, especially in trophoblast cells (a key component of the human placenta). In the hypoxia/reoxygenation (H/R)-exposed HTR-8/Svneo trophoblast cells, LAMP3 expression was also upregulated. H/R exposure repressed cell viability and invasion and increased apoptosis of trophoblast cells. siRNA-mediated knockdown of LAMP3 increased cell viability and invasion and suppressed apoptosis of H/R-exposed trophoblast cells. We further found that 1,25(OH)2D3 (the hormonally active form of vitamin D) treatment reduced LAMP3 expression in H/R exposed trophoblast cells. In addition, 1,25(OH)2D3 treatment promoted cell viability and invasion and inhibited apoptosis of H/R-exposed trophoblast cells. Notably, overexpression of LAMP3 abrogated the protective effect of 1,25(OH)2D3 on H/R-exposed trophoblast cells. Collectively, we demonstrated trophoblast cytoprotection by vitamin D, a process mediated via LAMP3.

ERα/ß/DMP1 axis promotes trans-differentiation of chondrocytes to bone cells through GSK-3ß/ß-catenin pathway.

Estrogen-induced premature closing of the growth plate in the long bones is a major cause of short stature after premature puberty. Recent studies have found that chondrocytes can directly trans-differentiate into osteoblasts in the process of endochondral bone formation, which indicates that cartilage formation and osteogenesis may be a continuous biological process. However, whether estrogen promotes the direct trans-differentiation of chondrocytes into osteoblasts remains largely unknown. Chondrocytes were treated with different concentrations of 17ß-estradiol, and Alizarin Red staining and alkaline phosphatase activity assay were used to detected osteogenesis. Specific short hairpin RNA and tamoxifen were used to block the estrogen receptor (ER) pathway and osteogenic marker genes and downstream gene expression were detected using real-time quantitative polymerase chain reaction, western blot, and immunohistochemistry staining. The findings showed that 17ß-estradiol promoted the chondrocyte osteogenesis in vitro, even at high concentrations. In addition, blocking of the ERα/ß pathway inhibited the trans-differentiation of chondrocytes into osteogenic cells. Furthermore, we found that dentin matrix protein 1 (DMP1), which is a direct downstream molecular of ER, was involved in 17ß-estradiol/ER pathway-regulated osteogenesis. As well, glycogen synthase kinase-3 beta (GSK-3ß)/ß-catenin signal pathway also participates in ERα/ß/DMP1-regulated chondrocyte osteogenesis. The GSK-3ß/ß-catenin signal pathway was involved in ERα/ß/DMP1-regulated chondrocyte osteogenesis. These findings suggest that ER/DMP1/GSK-3ß/ß-catenin plays a vital role in estrogen regulation of chondrocyte osteogenesis and provide a therapeutic target for short stature caused by epiphyseal fusion.

Vitamin D alleviates hypoxia/reoxygenation-induced injury of human trophoblast HTR-8 cells by activating autophagy.

INTRODUCTION: Attenuation of trophoblast cell dysfunction would be beneficial for retarding pre-eclampsia (PE). Vitamin D has been reported to improve trophoblast cell function in early PE, but the mechanism involved is not fully elucidated. This study is aimed to investigate whether vitamin D alleviates trophoblast cell dysfunction via regulating autophagy. METHODS: Human trophoblast HTR-8 cells were cultured in hypoxia/reoxygenation (H/R) condition to simulate the oxidative stress state of early PE in vitro. MTT, Transwell and tube formation assays were respectively applied to assess cell proliferation, invasion, and angiogenesis abilities. DCFH-DA staining was performed to detect cellular reactive oxygen species levels. GFP-RFP-LC3 plasmid transfection and transmission electron microscopy were subjected to monitor autophagy. Enzyme-linked immunosorbent assay and Western blot analysis were used to detect autophagy-related and pyroptosis-associated molecules. RESULTS: H/R led to severe impairments on the bio-function of HTR-8 cells, as evidenced by the deficiency of cell proliferation, invasion, and angiogenesis abilities, and the increase of cellular ROS production. Simultaneously, H/R inhibited autophagy and triggered pyroptosis. 1,25(OH)2D3, the hormonally active form of vitamin D, dramatically attenuated H/R-induced trophoblast dysfunction. Also, 1,25(OH)2D3 activated autophagy and inhibited pyroptosis. Additionally, autophagy-enhancer rapamycin exerted similar protective effect to that of 1,25(OH)2D3, whereas autophagy-inhibitor 3-methyladenine blocked the protective effect of 1,25(OH)2D3. DISCUSSION: The mechanism that vitamin D alleviates trophoblast cell dysfunction is associated with autophagy induction and pyroptosis inhibition.

A diagnostic model of idiopathic central precocious puberty based on transrectal pelvic ultrasound and basal gonadotropin levels.

OBJECTIVE: To establish a diagnostic model of idiopathic central precocious puberty on the basis of transrectal pelvic ultrasound and basal gonadotropin. METHODS: A total of 669 girls with Tanner breast development stage II were enrolled in this study from January 2015 to December 2018. The participants were divided into the ICPP group and the premature thelarche group. We analyzed various variables, including age at initial diagnosis, basal luteinizing hormone levels, the long diameter of the uterus, the transverse diameter of the uterus, the anterior-posterior diameter of the uterus, the volume of the uterus, maximum ovarian diameter, average ovarian volume, maximum ovarian volume, number of follicles (≥4 mm), maximum follicular diameter, endometrial thickness, and vaginal wall thickness. RESULTS: The following diagnostic model was established: Y=-14.123 + 0.630 × age at initial diagnosis + 1.119 × transverse diameter of the uterus + 1.278 × anterior-posterior diameter of the uterus + 0.637 × average ovarian volume + 1.316 × maximum ovarian diameter + 0.146 ×number of follicles ≥4 mm + 2.925 × endometrial thickness + 0.559 × basal luteinizing hormone value. The area under curve was 0.922, sensitivity was 84.9%, and specificity was 86.2%. CONCLUSION: Basal LH levels and transrectal pelvic ultrasound should be applied together to improve the accuracy of diagnosis in ICPP.

Methylmalonic Acidemia Complicated by Homocystinuria Diseases: a Report of Three Cases.

This study aims to improve our understanding of methylmalonic acidemia (MMA) complicated by homocystinuria disease by analyzing the clinical characteristics, treatment response and prognosis of three patients. Hyperhomocysteinemia and developmental retardation were present in all patients, epilepsy was present in one patient, and hemolytic uremic syndrome was present in one patient. The conditions of two patients were complicated by pulmonary arterial hypertension, one patient by left pulmonary vein ectopic drainage to the coronary sinus and the other by noncompaction of the ventricular myocardium. The two MMA patients with the complication of severe pulmonary arterial hypertension died because of late diagnosis and irregular treatment of MMA. Echocardiography is necessary for patients with combined MMA and homocystinuria, and these patients are susceptible to cardiovascular disease. When a patient with combined MMA and homocystinuria has the complication of severe pulmonary arterial hypertension, the prognosis is poor.

Novel genotypes and phenotypes among Chinese patients with Floating-Harbor syndrome.

BACKGROUND: Floating-Harbor syndrome (FHS) is a rare syndromic short stature disorder caused by truncating variants in SRCAP. Few Chinese FHS patients had been reported so far and limited knowledge regarding the benefit of growth hormone treatment existed. METHODS: We ascertained 12 short stature patients with molecularly confirmed diagnosis of FHS by whole exome sequencing. We performed a comprehensive clinical evaluation for all patients and assessed the responsiveness of growth hormone treatment in a subset of the patients. RESULTS: Five distinct pathogenic/likely pathogenic variants were identified in 12 independent FHS patients including two previously reported variants (c.7303C > T/p.Arg2435Ter and c.7330C > T/p.Arg2444Ter) and three novel variants (c.7189G > T/p.Glu2397Ter, c.7245_7246delAT/p.Ser2416ArgfsTer26 and c.7466C > G/p.Ser2489Ter). The c.7303C > T/p.Arg2435Ter mutation appears more common in Chinese FHS patients. The clinical presentations of Chinese FHS patients are very similar to those of previously reported patients of different ethnicities. Yet we noticed micropenis and ear abnormalities in multiple patients, suggesting that these may be novel phenotypes of Floating-Harbor syndrome. Eight patients (one with GH deficiency, one with undetermined GH level, six without GH deficiency) underwent growth hormone treatment, 3 patients had good responses, one with modest and two with poor responses. CONCLUSION: We described novel genotypes and phenotypes in a Chinese FHS patient cohort. We showed that about half of FHS patients exhibited modest to good response to GH treatment regardless of their respective GH deficiency status. We didn't find any correlation between different mutations and response to GH treatment.

[Clinical and mutational analysis of 7 children with X-linked adrenal dysplasia congenita].

OBJECTIVE: To summarize clinical manifestations, inheritance pattern and mutations of NR0B1 gene in 7 children with X-linked adrenal dysplasia congenita (XL-AHC). METHODS: Clinical data of the 7 children was collected. Next-generation sequencing was carried out to detect potential mutations in the coding regions of adrenal gland-related genes. Suspected mutations were verified with Sanger sequencing. RESULTS: In all of the children, the initial symptom was adrenocortical insufficiency. Five cases had neonatal onset, while the remaining two developed it at the age of 2. Three cases (42.9%) had a short stature and 1 showed growth retardation (14.3%). Of the 7 cases, 6 (85.7%) had mutations occurring in exon 1, and 1 (14.3%) had it occurring in exon 2. Four cases (57.1%) were frameshift mutations, 2 cases (28.6%) were nonsense mutations and 1 case (14.3%) was missense mutation. Two mutations were known to be pathogenic, and 5 had not been reported previously. Maternal inheritance was found in 6 cases. Three children had a maternal uncle died of unexplained causes. The mothers of 2 children had a history of spontaneous abortions. One child had a brother died of unexplained reason. CONCLUSION: Male children with primary adrenal insufficiency should be routinely checked for NR0B1 mutations, especially those with a family history. mutations of NR0B1 gene occur mostly in exon 1, with frameshift mutations being the most common type. The development of all patients with XL-AHC should be closely monitored during follow-up.

[Clinical features and genetic analysis of seven patients with congenital hyperinsulinism].

OBJECTIVE: To analyze clinical characteristics, genetic mutation and therapeutic effect of seven patients diagnosed with congenital hyperinsulinism(CHI). METHODS: Clinical data for the patients was retrospectively analyzed. RESULTS: All patients presented with hyperinsulinism(serum insulin:2.0-58.4 mU/L),even after hypoglycemia (blood glucose: 0.7-2.39 mmol/L) has developed. Mutations were identified in 4 patients (57.1%), which included a heterozygous c.262C to T(p.R88C) mutation in exon 4 of the UCP2 gene, a heterozygous c.1495C to A(p.G499C) mutation in exon 12 of the GLUD1 gene, a heterozygous c.1493C to T(p.S498L) mutation in exon 1 of the GLUD1 gene, and a heterozygous c.4432G to A(p.G1478R) mutation in exon 37 of the ABCC8 gene. The patient carrying a maternally inherited ABCC8 mutation was treated with cornstarch and had his blood glucose kept normal. All other patients responded well to diazoxide. CONCLUSION: A genetic diagnosis was attained for 51.7% of patients in this study. Mild CHI patients can have their blood glucose controlled by giving cornstarch. Diazoxide is safe and effective for most CHI patients.

[Dexamethasone up-regulates the expression of glucocorticoid receptor in growth plate and inhibits the longitudinal growth of bone: experiment with rats].

OBJECTIVE: To investigate the regulation of the glucocorticoid of pharmacological doses on the expression of glucocorticoid receptor in growth plate and to explore the mechanism of the direct inhibition of the longitudinal growth of bone by dexamethasone. METHODS: Twenty 3-week-old male weanling SD rats were randomly divided into two groups: dexamethasone group (n = 12), intraperitoneally injected with dexamethasone 200 microg/100 g body weight once a day for 10 days and control group (n = 8), intraperitoneally injected with normal saline of the same volume. Ten days later the rats were killed. The length of the proximal tibia was excised, fixed and decalcified, and then paraffin embedded. Sections of 5 microm thick were cut and processed for histomorphometry The total height of growth plate, height of proliferative zone, and height of hypertrophic zone were determined. RESULTS: The length of tibia, total height of growth plate, height of proliferative zone, and height of hypertrophic zone of the dexamethasone group were 28.9 mm +/- 1.2 mm, 4.01 microm +/- 0.28 microm, 1.98 microm +/- 0.13 microm, and 1.67 microm +/- 0.18 microm respectively, all significantly lower those of the control group (30.5 mm +/- 1.1 mm, 5.53 microm +/- 0.46 microm, 2.25 microm +/- 0.30 microm, and 2.87 microm +/- 0.19 microm respectively, all P < 0.01). The resting chondrocytes and hypertrophic chondrocytes in the growth plates of the rats in the dexamethasone group all expressed glucocorticoid receptor, whereas the proliferating chondrocytes didn't. The absorbance values of the resting chondrocytes and hypertrophic chondrocytes in the growth plates of the dexamethasone group were 0.238 +/- 0.026 and 0.283 +/- 0.042 respectively, both significantly higher than those of the control group (0.187 +/- 0.027 and 0.211 +/- 0.022 respectively, both P < 0.01). CONCLUSION: The glucocorticoid receptors are essential for the longitudinal growth of bone. The glucocorticoid of pharmacological dose inhibits the longitudinal growth of bone by up-regulating the expression of glucocorticoid receptor.