Site-1 protease is essential for endochondral bone formation in mice

Site-1 protease (S1P) has an essential function in the conversion of latent, membrane-bound transcription factors to their free, active form. In mammals, abundant expression of S1P in chondrocytes suggests an involvement in chondrocyte function. To determine the requirement of S1P in cartilage and bone development, we have created cartilage-specific S1P knockout mice (S1P(cko)). S1P(cko) mice exhibit chondrodysplasia and a complete lack of endochondral ossification even though Runx2 expression, Indian hedgehog signaling, and osteoblastogenesis is intact. However, there is a substantial increase in chondrocyte apoptosis in the cartilage of S1P(cko) mice. Extraction of type II collagen is substantially lower from S1P(cko) cartilage. In S1P(cko) mice, the collagen network is disorganized and collagen becomes entrapped in chondrocytes. Ultrastructural analysis reveals that the endoplasmic reticulum (ER) in S1P(cko) chondrocytes is engorged and fragmented in a manner characteristic of severe ER stress. These data suggest that S1P activity is necessary for a specialized ER stress response required by chondrocytes for the genesis of normal cartilage and thus endochondral ossification.

Biological mediators and periodontal regeneration: a review of enamel matrix proteins at the cellular and molecular levels

BACKGROUND: Despite a large body of clinical and histological data demonstrating beneficial effects of enamel matrix proteins (EMPs) for regenerative periodontal therapy, it is less clear how the available biological data can explain the mechanisms underlying the supportive effects of EMPs. OBJECTIVE: To analyse all available biological data of EMPs at the cellular and molecular levels that are relevant in the context of periodontal wound healing and tissue formation. METHODS: A stringent systematic approach was applied using the key words "enamel matrix proteins" OR "enamel matrix derivative" OR "emdogain" OR "amelogenin". The literature search was performed separately for epithelial cells, gingival fibroblasts, periodontal ligament cells, cementoblasts, osteogenic/chondrogenic/bone marrow cells, wound healing, and bacteria. RESULTS: A total of 103 papers met the inclusion criteria. EMPs affect many different cell types. Overall, the available data show that EMPs have effects on: (1) cell attachment, spreading, and chemotaxis; (2) cell proliferation and survival; (3) expression of transcription factors; (4) expression of growth factors, cytokines, extracellular matrix constituents, and other macromolecules; and (5) expression of molecules involved in the regulation of bone remodelling. CONCLUSION: All together, the data analysis provides strong evidence for EMPs to support wound healing and new periodontal tissue formation.

Adrenal gland development and defects

The network regulating human adrenal development is complex. Studies of patients with adrenal insufficiency due to gene mutations established a central role for transcription factors GLI3, SF1 and DAX1 in the initial steps of adrenal formation. Adrenal differentiation seems to depend on adrenocorticotropic hormone (ACTH) stimulation and signalling, including biosynthesis and action of POMC, PC1, TPIT, MC2R, MRAP and ALADIN, all of which cause adrenocortical hypoplasia when mutated in humans. Studies of knockout mice revealed many more factors involved in adrenal development; however, in contrast to rodents, in humans several of those factors had no adrenal phenotype when mutated (e.g. WT1, WNT4) or, alternatively, human mutations have not (yet) been identified. Tissue profiling of fetal and adult adrenals suggested 69 genes involved in adrenal development. Among them were genes coding for steroidogenic enzymes, transcription and growth factors, signalling molecules, regulators of cell cycle and angiogenesis, and extracellular matrix proteins; however, the exact role of most of them remains to be elucidated.

Adipogenic and myogenic gene expression in rotator cuff muscle of the sheep after tendon tear

Chronic rotator cuff tendon tears lead to fatty infiltration and muscle atrophy with impaired physiological functions of the affected muscles. However, the cellular and molecular mechanisms of corresponding pathophysiological processes remain unknown. The purpose of this study was to characterize the expression pattern of adipogenic (PPARgamma, C/EBPbeta) and myogenic (myostatin, myogenin, Myf-5) transcription factors in infraspinatus muscle of sheep after tenotomy, implantation of a tension device, refixation of the tendon, and rehabilitation, reflecting a model of chronic rotator cuff tears. In contrast to human patients, the presented sheep model allows a temporal evaluation of the expression of a given marker in the same individual over time. Semiquantitative RT/PCR analysis of PPARgammaã, myostatin, myogenin, Myf-5, and C/EBPbeta transcript levels was carried out with sheep muscle biopsy-derived total RNA. We found a significantly increased expression of Myf-5 and PPARgamma after tenotomy and a significant change for Myf-5 and C/EBPbeta after continuous traction and refixation. This experimental sheep model allows the molecular analysis of pathomechanisms of muscular changes after rotator cuff tear. The results point to a crucial role of the transcription factors PPARgamma, C/EBPbeta, and Myf-5 in impairment and regeneration of rotator cuff muscles after tendon tears in sheep.

The tumor suppressor gene hypermethylated in cancer 1 is transcriptionally regulated by E2F1

The Hypermethylated in Cancer 1 (HIC1) gene encodes a zinc finger transcriptional repressor that cooperates with p53 to suppress cancer development. We and others recently showed that HIC1 is a transcriptional target of p53. To identify additional transcriptional regulators of HIC1, we screened a set of transcription factors for regulation of a human HIC1 promoter reporter. We found that E2F1 strongly activates the full-length HIC1 promoter reporter. Promoter deletions and mutations identified two E2F responsive elements in the HIC1 core promoter region. Moreover, in vivo binding of E2F1 to the HIC1 promoter was shown by chromatin immunoprecipitation assays in human TIG3 fibroblasts expressing tamoxifen-activated E2F1. In agreement, activation of E2F1 in TIG3-E2F1 cells markedly increased HIC1 expression. Interestingly, expression of E2F1 in the p53(-/-) hepatocellular carcinoma cell line Hep3B led to an increase of endogenous HIC1 mRNA, although bisulfite genomic sequencing of the HIC1 promoter revealed that the region bearing the two E2F1 binding sites is hypermethylated. In addition, endogenous E2F1 induced by etoposide treatment bound to the HIC1 promoter. Moreover, inhibition of E2F1 strongly reduced the expression of etoposide-induced HIC1. In conclusion, we identified HIC1 as novel E2F1 transcriptional target in DNA damage responses.

Mechanistic and therapeutic implications of angiogenesis in endometriosis

Like tumor metastases, endometriotic implants require neovascularization to proliferate and invade into ectopic sites within the host. Endometrial tissue, with its robust stem cell populations and remarkable regenerative capabilities, is a rich source of proangiogenic factors. Among the most potent and extensively studied of these proteins, vascular endothelial growth factor has emerged as a critical vasculogenic regulator in endometriosis. Accordingly, angiogenesis of the nascent endometriotic lesion has become an attractive target for novel medical therapeutics and strategies to inhibit vascular endothelial growth factor action. Vascular endothelial growth factor gene regulation in endometrial and endometriosis cells by nuclear receptors, other transcription factors, and also by infiltrating immune cells is emphasized. New data showing that oxidative and endoplasmic reticulum stress increase vascular endothelial growth factor expression are provided. Finally, we review the clinical implications of angiogenesis in this condition and propose potential antiangiogenic therapies that may become useful in the control or eradication of endometriotic lesions.

Role of DNA methylation in the tissue-specific expression of the CYP17A1 gene for steroidogenesis in rodents

The CYP17A1 gene is the qualitative regulator of steroidogenesis. Depending on the presence or absence of CYP17 activities mineralocorticoids, glucocorticoids or adrenal androgens are produced. The expression of the CYP17A1 gene is tissue as well as species-specific. In contrast to humans, adrenals of rodents do not express the CYP17A1 gene and have therefore no P450c17 enzyme for cortisol production, but produce corticosterone. DNA methylation is involved in the tissue-specific silencing of the CYP17A1 gene in human placental JEG-3 cells. We investigated the role of DNA methylation for the tissue-specific expression of the CYP17A1 gene in rodents. Rats treated with the methyltransferase inhibitor 5-aza-deoxycytidine excreted the cortisol metabolite tetrahydrocortisol in their urine suggesting that treatment induced CYP17 expression and 17alpha-hydroxylase activity through demethylation. Accordingly, bisulfite modification experiments identified a methylated CpG island in the CYP17 promoter in DNA extracted from rat adrenals but not from testes. Both methyltransferase and histone deacetylase inhibitors induced the expression of the CYP17A1 gene in mouse adrenocortical Y1 cells which normally do not express CYP17, indicating that the expression of the mouse CYP17A1 gene is epigenetically controlled. The role of DNA methylation for CYP17 expression was further underlined by the finding that a reporter construct driven by the mouse -1041 bp CYP17 promoter was active in Y1 cells, thus excluding the lack of essential transcription factors for CYP17 expression in these adrenal cells.

Differential expression and localization of lipid transporters in the bovine mammary gland during the pregnancy-lactation cycle

The transport of lipids across mammary gland epithelial cells (MEC) determines milk lipid content and composition. We investigated the expression of lipid transporters and their regulators in comparison to blood metabolites during lactation and dry period (DP) in dairy cows. Repeated mammary gland biopsies and blood samples were taken from 10 animals at 7 stages of the pregnancy-lactation cycle. Expression levels of the specific mRNAs were determined by quantitative reverse transcription-PCR, whereas ABCA1 was localized by immunohistochemistry. Blood serum metabolites were determined by common enzymatic chemistries. Elevated mRNA profiles of ABCA1 and ABCA7 were found during DP as compared with lactation and were inversely associated with blood cholesterol levels. Elevated levels of ABCG2, NPC1, SREBP1, SREBP2, LXR alpha, and PPAR gamma were found postpartum, whereas ABCG1 did not differ between the functional stages of the mammary gland. The ABCA1 protein was localized in MEC and showed differential activity between DP and lactation suggesting a role of ABCA1 in the removal of excess cellular cholesterol from MEC during the DP. The expression profiles of ABCA7 and NPC1 may reflect a role of these transporters in the clearance of apoptotic cells and the intracellular redistribution of cholesterol, respectively. Regulation of lipid transporters in the mammary gland is partially associated with transcription factors that control lipid homeostasis.

Cardiac c-kit+AT2+ cell population is increased in response to ischemic injury and supports cardiomyocyte performance

The expression pattern of angiotensin AT2 receptors with predominance during fetal life and upregulation under pathological conditions during tissue injury/repair process suggests that AT2 receptors may exert an important action in injury/repair adaptive mechanisms. Less is known about AT2 receptors in acute ischemia-induced cardiac injury. We aimed here to elucidate the role of AT2 receptors after acute myocardial infarction. Double immunofluorescence staining showed that cardiac AT2 receptors were mainly detected in clusters of small c-kit+ cells accumulating in peri-infarct zone and c-kit+AT2+ cells increased in response to acute cardiac injury. Further, we isolated cardiac c-kit+AT2+ cell population by modified magnetic activated cell sorting and fluorescence activated cell sorting. These cardiac c-kit+AT2+ cells, represented approximately 0.19% of total cardiac cells in infarcted heart, were characterized by upregulated transcription factors implicated in cardiogenic differentiation (Gata-4, Notch-2, Nkx-2.5) and genes required for self-renewal (Tbx-3, c-Myc, Akt). When adult cardiomyocytes and cardiac c-kit+AT2+ cells isolated from infarcted rat hearts were cocultured, AT2 receptor stimulation in vitro inhibited apoptosis of these cocultured cardiomyocytes. Moreover, in vivo AT2 receptor stimulation led to an increased c-kit+AT2+ cell population in the infarcted myocardium and reduced apoptosis of cardiomyocytes in rats with acute myocardial infarction. These data suggest that cardiac c-kit+AT2+ cell population exists and increases after acute ischemic injury. AT2 receptor activation supports performance of cardiomyocytes, thus contributing to cardioprotection via cardiac c-kit+AT2+ cell population.

Variation in hepatic regulation of metabolism during the dry period and in early lactation in dairy cows

The purpose of this study was to investigate variations in hepatic regulation of metabolism during the dry period, after parturition, and in early lactation in dairy cows. For this evaluation, cows were divided into 2 groups based on the plasma concentration of beta-hydroxybutyric acid (BHBA) in wk 4 postpartum (PP; group HB, BHBA >0.75 mmol/L; group LB, BHBA <0.75 mmol/L, respectively). Liver biopsies were obtained from 28 cows at drying off (mean 59 +/- 8 d antepartum), on d 1, and in wk 4 and 14 PP. Blood samples were collected every 2 wk during this entire period. Liver samples were analyzed for mRNA abundance of genes related to carbohydrate metabolism (pyruvate carboxylase, PC; phosphoenolpyruvate carboxykinase, PEPCK; citrate synthase, CS), fatty acid biosynthesis (ATP citrate lyase, ACLY) and oxidation (acyl-CoA synthetase long-chain, ACSL; carnitine palmitoyltransferase 1A, CPT 1A; carnitine palmitoyltransferase 2, CPT 2; acyl-coenzyme A dehydrogenase very long chain, ACADVL), cholesterol biosynthesis (3-hydroxy-3-methylglutaryl-coenzyme A synthase 1, HMGCS1), ketogenesis (3-hydroxy-3-methylglutaryl-coenzyme A synthase 2, HMGCS2), and of genes encoding the transcription factors peroxisome proliferator-activated receptor alpha (PPARalpha), peroxisome proliferator-activated receptor gamma (PPARgamma), and sterol regulatory element binding factor 1 (SREBF1). Blood plasma was assayed for concentrations of glucose, BHBA, nonesterified fatty acids, cholesterol, triglycerides, insulin, insulin-like growth factor-I, and thyroid hormones. In both groups, plasma parameters followed a pattern usually observed in dairy cows. However, changes were moderate and the energy balance in cows turned positive in wk 7 PP for both groups. Additionally, the energy balance and milk yield were similar for both groups after parturition onwards. Significant group effects were found at drying off, when plasma concentrations of triglycerides were higher in LB than in HB, and in wk 4 PP, when plasma concentrations of glucose and IGF-I were lower in HB than in LB. Similarly, moderate changes in mRNA expression of hepatic genes between the different time points were observed, although HB cows showed more adaptive performance than LB cows based on changes in mRNA expression of PEPCKc, PEPCKm, CS, CPT 1A, CPT 2, and PPARalpha. Part of the variation measured in this study was explained by parity. Significant Spearman rank correlation coefficients between the variables were not similar at each time point and were not similar between the groups at each time point, suggesting that metabolic regulation differs between cows. In conclusion, metabolic regulation in dairy cows is a dynamic system, and differs obviously between cows at different metabolic stages related to parturition.

Gene array of primary human osteoblasts exposed to enamel matrix derivative in combination with a natural bone mineral

OBJECTIVES The application of an enamel matrix derivative (EMD) for regenerative periodontal surgery has been shown to promote formation of new cementum, periodontal ligament, and alveolar bone. In intrabony defects with a complicated anatomy, the combination of EMD with various bone grafting materials has resulted in additional clinical improvements, but the initial cellular response of osteoblasts coming in contact with these particles have not yet been fully elucidated. The objective of the present study was to evaluate the in vitro effects of EMD combined with a natural bone mineral (NBM) on a wide variety of genes, cytokines, and transcription factors and extracellular matrix proteins on primary human osteoblasts. MATERIAL AND METHODS Primary human osteoblasts were seeded on NBM particles pre-coated with versus without EMD and analyzed for gene differences using a human osteogenesis gene super-array (Applied Biosystems). Osteoblast-related genes include those transcribed during bone mineralization, ossification, bone metabolism, cell growth and differentiation, as well as gene products representing extracellular matrix molecules, transcription factors, and cell adhesion molecules. RESULTS EMD promoted gene expression of various osteoblast differentiation markers including a number of collagen types and isoforms, SMAD intracellular proteins, osteopontin, cadherin, alkaline phosphatase, and bone sialoprotein. EMD also upregulated a variety of growth factors including bone morphogenetic proteins, vascular endothelial growth factors, insulin-like growth factor, transforming growth factor, and their associated receptor proteins. CONCLUSION The results from the present study demonstrate that EMD is capable of activating a wide variety of genes, growth factors, and cytokines when pre-coated onto NBM particles. CLINICAL RELEVANCE The described in vitro effects of EMD on human primary osteoblasts provide further biologic support for the clinical application of a combination of EMD with NBM particles in periodontal and oral regenerative surgery.

Skeletal muscle mitochondria in the elderly: effects of physical fitness and exercise training.

Context: Sarcopenia is thought to be associated with mitochondrial (M) loss. It is unclear whether the decrease in M content is consequent to aging per se or to decreased physical activity. Objectives: To examine the influence of fitness on M content and function, and to assess whether exercise could improve M function in older adults. Design and subjects: Three distinct studies were conducted: 1) a cross-sectional observation comparing M content and fitness in a large heterogeneous cohort of older adults; 2) a case-control study comparing chronically endurance-trained older adults (A) and sedentary (S) subjects matched for age and gender; 3) a 4-month exercise intervention in S. Setting: University-based clinical research center Outcomes: M volume density (Mv) was assessed by electron microscopy from vastus lateralis biopsies, electron transport chain proteins (ETC) by western blotting, mRNAs for transcription factors involved in M biogenesis by qRT-PCR and in-vivo oxidative capacity (ATPmax) by (31)P-MR spectroscopy. Peak oxygen uptake (VO2peak) was measured by GXT. Results: VO2peak was strongly correlated with Mv in eighty 60-80 yo adults. Comparison of A vs. S revealed differences in Mv, ATPmax and some ETC complexes. Finally, exercise intervention confirmed that S are able to recover Mv, ATPmax and specific transcription factors. Conclusions: These data suggest that 1) aging per se is not the primary culprit leading to M dysfunction, 2) an aerobic exercise program, even at an older age, can ameliorate the loss in skeletal muscle M content and may prevent aging muscle comorbidities and 3) the improvement of M function is all about content.

Hepatic gene expression involved in glucose and lipid metabolism in transition cows: effects of fat mobilization during early lactation in relation to milk performance and metabolic changes.

Insufficient feed intake during early lactation results in elevated body fat mobilization to meet energy demands for milk production. Hepatic energy metabolism is involved by increasing endogenous glucose production and hepatic glucose output for milk synthesis and by adaptation of postcalving fuel oxidation. Given that cows differ in their degree of fat mobilization around parturition, indicated by variable total liver fat concentration (LFC), the study investigated the influence of peripartum fat mobilization on hepatic gene expression involved in gluconeogenesis, fatty acid oxidation, ketogenesis, and cholesterol synthesis, as well as transcriptional factors referring to energy metabolism. German Holstein cows were grouped according to mean total LFC on d 1, 14, and 28 after parturition as low [<200mg of total fat/g of dry matter (DM); n=10], medium (200-300 mg of total fat/g of DM; n=10), and high (>300 mg of total fat/g of DM; n=7), indicating fat mobilization during early lactation. Cows were fed total mixed rations ad libitum and held under equal conditions. Liver biopsies were taken at d 56 and 15 before and d 1, 14, 28, and 49 after parturition to measure mRNA abundances of pyruvate carboxylase (PC); phosphoenolpyruvate carboxykinase; glucose-6-phosphatase; propionyl-coenzyme A (CoA) carboxylase α; carnitine palmitoyl-transferase 1A (CPT1A); acyl-CoA synthetase, long chain 1 (ASCL1); acyl-CoA dehydrogenase, very long chain; 3-hydroxy-3-methylglutaryl-CoA synthase 1 and 2; sterol regulatory element-binding factor 1; and peroxisome proliferator-activated factor α. Total LFC postpartum differed greatly among cows, and the mRNA abundance of most enzymes and transcription factors changed with time during the experimental period. Abundance of PC mRNA increased at parturition to a greater extent in high- and medium-LFC groups than in the low-LFC group. Significant LFC × time interactions for ACSL1 and CPT1A during the experimental period indicated variable gene expression depending on LFC after parturition. Correlations between hepatic gene expression and performance data and plasma concentrations of metabolites and hormones showed time-specific relations during the transition period. Elevated body fat mobilization during early lactation affected gene expression involved in gluconeogenesis to a greater extent than gene expression involved in lipid metabolism, indicating the dependence of hepatic glucose metabolism on hepatic lipid status and fat mobilization during early lactation.

Differential regulation of human 3β-hydroxysteroid dehydrogenase type 2 for steroid hormone biosynthesis by starvation and cyclic AMP stimulation: studies in the human adrenal NCI-H295R cell model

Human steroid biosynthesis depends on a specifically regulated cascade of enzymes including 3β-hydroxysteroid dehydrogenases (HSD3Bs). Type 2 HSD3B catalyzes the conversion of pregnenolone, 17α-hydroxypregnenolone and dehydroepiandrosterone to progesterone, 17α-hydroxyprogesterone and androstenedione in the human adrenal cortex and the gonads but the exact regulation of this enzyme is unknown. Therefore, specific downregulation of HSD3B2 at adrenarche around age 6-8 years and characteristic upregulation of HSD3B2 in the ovaries of women suffering from the polycystic ovary syndrome remain unexplained prompting us to study the regulation of HSD3B2 in adrenal NCI-H295R cells. Our studies confirm that the HSD3B2 promoter is regulated by transcription factors GATA, Nur77 and SF1/LRH1 in concert and that the NBRE/Nur77 site is crucial for hormonal stimulation with cAMP. In fact, these three transcription factors together were able to transactivate the HSD3B2 promoter in placental JEG3 cells which normally do not express HSD3B2. By contrast, epigenetic mechanisms such as methylation and acetylation seem not involved in controlling HSD3B2 expression. Cyclic AMP was found to exert differential effects on HSD3B2 when comparing short (acute) versus long-term (chronic) stimulation. Short cAMP stimulation inhibited HSD3B2 activity directly possibly due to regulation at co-factor or substrate level or posttranslational modification of the protein. Long cAMP stimulation attenuated HSD3B2 inhibition and increased HSD3B2 expression through transcriptional regulation. Although PKA and MAPK pathways are obvious candidates for possibly transmitting the cAMP signal to HSD3B2, our studies using PKA and MEK1/2 inhibitors revealed no such downstream signaling of cAMP. However, both signaling pathways were clearly regulating HSD3B2 expression.

"Hot spot" in the PROP1 gene responsible for combined pituitary hormone deficiency.

As pituitary function depends on the integrity of the hypothalamic-pituitary axis, any defect in the development and organogenesis of this gland may account for a form of combined pituitary hormone deficiency (CPHD). Although pit-1 was 1 of the first factors identified as a cause of CPHD in mice, many other homeodomain and transcription factors have been characterized as being involved in different developmental stages of pituitary gland development, such as prophet of pit-1 (prop-1), P-Lim, ETS-1, and Brn 4. The aims of the present study were first to screen families and patients suffering from different forms of CPHD for PROP1 gene alterations, and second to define possible hot spots and the frequency of the different gene alterations found. Of 73 subjects (36 families) analyzed, we found 35 patients, belonging to 18 unrelated families, with CPHD caused by a PROP1 gene defect. The PROP1 gene alterations included 3 missense mutations, 2 frameshift mutations, and 1 splice site mutation. The 2 reported frameshift mutations could be caused by any 2-bp GA or AG deletion at either the 148-GGA-GGG-153 or 295-CGA-GAG-AGT-303 position. As any combination of a GA or AG deletion yields the same sequencing data, the frameshift mutations were called 149delGA and 296delGA, respectively. All but 1 mutation were located in the PROP1 gene encoding the homeodomain. Importantly, 3 tandem repeats of the dinucleotides GA at location 296-302 in the PROP1 gene represent a hot spot for CPHD. In conclusion, the PROP1 gene seems to be a major candidate gene for CPHD; however, further studies are needed to evaluate other genetic defects involved in pituitary development.