NAADP-binding proteins find their identity.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a second messenger that releases Ca2+ from endosomes and lysosomes by activating ion channels called two-pore channels (TPCs). However, no NAADP-binding site has been identified on TPCs. Rather, NAADP activates TPCs indirectly by engaging NAADP-binding proteins (NAADP-BPs) that form part of the TPC complex. After a decade of searching, two different NAADP-BPs were recently identified: Jupiter microtubule associated homolog 2 (JPT2) and like-Sm protein 12 (LSM12). These discoveries bridge the gap between NAADP generation and NAADP activation of TPCs, providing new opportunity to understand and manipulate the NAADP-signaling pathway. The unmasking of these NAADP-BPs will catalyze future studies to define the molecular choreography of NAADP action.

Regulating the cell shift of endothelial cell-like myofibroblasts in pulmonary fibrosis.

Pulmonary fibrosis is a common and severe fibrotic lung disease with high morbidity and mortality. Recent studies have reported a large number of unwanted myofibroblasts appearing in pulmonary fibrosis, and shown that the sustained activation of myofibroblasts is essential for unremitting interstitial fibrogenesis. However, the origin of these myofibroblasts remains poorly understood. Here, we create new mouse models of pulmonary fibrosis and identify a previously unknown population of endothelial cell (EC)-like myofibroblasts in normal lung tissue. We show that these EC-like myofibroblasts significantly contribute myofibroblasts to pulmonary fibrosis, which is confirmed by single-cell RNA sequencing of human pulmonary fibrosis. Using the transcriptional profiles, we identified a small molecule that redirects the differentiation of EC-like myofibroblasts and reduces pulmonary fibrosis in our mouse models. Our study reveals the mechanistic underpinnings of the differentiation of EC-like myofibroblasts in pulmonary fibrosis and may provide new strategies for therapeutic interventions.

Single-Cell RNA-Seq Identifies Dynamic Cardiac Transition Program from ADCs Induced by Leukemia Inhibitory Factor.

Adipose-derived cells (ADCs) from white adipose tissue are promising stem cell candidates because of their large regenerative reserves and the potential for cardiac regeneration. However, given the heterogeneity of ADC and its unsolved mechanisms of cardiac acquisition, ADC-cardiac transition efficiency remains low. In this study, we explored the heterogeneity of ADCs and the cellular kinetics of 39,432 single-cell transcriptomes along the leukemia inhibitory factor (LIF)-induced ADC-cardiac transition. We identified distinct ADC subpopulations that reacted differentially to LIF when entering the cardiomyogenic program, further demonstrating that ADC-myogenesis is time-dependent and initiates from transient changes in nuclear factor erythroid 2-related factor 2 (Nrf2) signaling. At later stages, pseudotime analysis of ADCs navigated a trajectory with 2 branches corresponding to activated myofibroblast or cardiomyocyte-like cells. Our findings offer a high-resolution dissection of ADC heterogeneity and cell fate during ADC-cardiac transition, thus providing new insights into potential cardiac stem cells.

GSK3ß Inhibition Reduced Vascular Calcification in Ins2Akita/+ Mice.

Endothelial-mesenchymal transition (EndMT) drives the endothelium to contribute to vascular calcification in diabetes mellitus. In our previous study, we showed that glycogen synthase kinase-3ß (GSK3ß) inhibition induces ß-catenin and reduces mothers against DPP homolog 1 (SMAD1) to direct osteoblast-like cells toward endothelial lineage, thereby reducing vascular calcification in Matrix Gla Protein (Mgp) deficiency. Here, we report that GSK3ß inhibition reduces vascular calcification in diabetic Ins2Akita/wt mice. Cell lineage tracing reveals that GSK3ß inhibition redirects endothelial cell (EC)-derived osteoblast-like cells back to endothelial lineage in the diabetic endothelium of Ins2Akita/wt mice. We also find that the alterations in ß-catenin and SMAD1 by GSK3ß inhibition in the aortic endothelium of diabetic Ins2Akita/wt mice are similar to Mgp-/- mice. Together, our results suggest that GSK3ß inhibition reduces vascular calcification in diabetic arteries through a similar mechanism to that in Mgp-/- mice.

GSK3ß Inhibition Ameliorates Atherosclerotic Calcification.

Endothelial-mesenchymal transition (EndMT) drives endothelium to contribute to atherosclerotic calcification. In a previous study, we showed that glycogen synthase kinase-3ß (GSK3ß) inhibition induced ß-catenin and reduced mothers against DPP homolog 1 (SMAD1) in order to redirect osteoblast-like cells towards endothelial lineage, thereby reducing vascular calcification in Matrix Gla Protein (Mgp) deficiency and diabetic Ins2Akita/wt mice. Here, we report that GSK3ß inhibition or endothelial-specific deletion of GSK3ß reduces atherosclerotic calcification. We also find that alterations in ß-catenin and SMAD1 induced by GSK3ß inhibition in the aortas of Apoe-/- mice are similar to Mgp-/- mice. Together, our results suggest that GSK3ß inhibition reduces vascular calcification in atherosclerotic lesions through a similar mechanism to that in Mgp-/- mice.

Intact parathyroid hormone levels and primary hyperparathyroidism.

OBJECTIVE: The aim of this article is to compare clinical characteristics and lab values for metabolic syndrome between primary hyperparathyroidism (PHPT) patients with different levels of serum intact parathyroid hormone (iPTH) and to determine correlation between different clinical characteristics among PHPT patients Methods: We reviewed charts of 212 PHPT patients in this retrospective study. Patients were divided into two groups according to their initial serum iPTH levels. Student's t-tests were used to compare the two groups for differences in clinical characteristics and laboratory values. Pearson's correlation coefficients were used to assess associations. RESULTS: Of the 212 PHPT patients, 100 were classified as m-iPTH group (serum iPTH < 140 pg/mL), whereas 112 patients were defined as h-iPTH group (serum iPTH ≥ 140 pg/mL). The h-iPTH patients were younger, had higher serum calcium and alkaline phosphatase levels, but exhibited lower 25(OH)-vitamin D and HDL levels, when compared with those of m-iPTH patients. Adenoma weights in the h-iPTH group tended to be higher than that in the m-iPTH group. Furthermore, association studies revealed that the iPTH level was positively correlated with adenoma weight and serum calcium and triglyceride (TG) levels but negatively correlated with HDL level. CONCLUSION: Our study supports the hypothesis that iPTH level is associated with TG and HDL levels and should be a factor to consider in the management of PHPT patients.

IMPACT OF ETHNIC BACKGROUND ON CLINICAL CHARACTERISTICS AND CARDIOVASCULAR RISK FACTORS AMONG PATIENTS WITH PRIMARY HYPERPARATHYROIDISM.

OBJECTIVE: To compare initial laboratory values and cardiovascular risk factors (CRF) among patients with primary hyperparathyroidism (PHPT) of different ethnic backgrounds. METHODS: In this retrospective study, we reviewed 500 charts of PHPT patients who presented at Robert Wood Johnson University Hospital from January 2000 to December 2013. Among these patients were 46 African Americans (AA), 31 Asians (A), 19 Hispanics (H), and 404 Caucasians (C). The following characteristics were compared between the groups: age; body mass index (BMI); levels of serum calcium, intact parathyroid hormone (iPTH), 25-OH vitamin D, and 24-hour urine calcium; and parathyroid adenoma weight. Presence of CRF including BMI, diabetes mellitus, hypertension, and hyperlipidemia were also recorded for comparison. Associations of adenoma weight and several other parameters were also assessed. RESULTS: Among different ethnic groups, AA patients with PHPT had higher iPTH levels compared to the A and C groups (P<.05), while 25-OHD levels were lower in the AA compared to the A and C groups (P<.05). Adenoma weight was significantly greater in AA than in C and A PHPT patients (P<.01). Adenoma weight was positively correlated with iPTH levels (r = 0.493, P <.001) and serum calcium levels (r = 0.255, P<.01). The group BMIs were C: 29.5 ± 6.9, AA: 33.8 ± 10, A: 24.7 ± 3.3, and H: 30.2 ± 6.6. AA patients had a lower rate of renal stones (9%) compared to other groups (21-29%, P<.05). CONCLUSION: The results of our study indicate that AA patients with PHPT presented with a more severe PHPT profile but had lower 24-hour urine calcium and fewer renal stones. AA patients with PHPT also had higher prevalence of CRF when compared to A and C.

Early evolution of the eukaryotic Ca2+ signaling machinery: conservation of the CatSper channel complex.

Calcium signaling is one of the most extensively employed signal transduction mechanisms in life. As life evolved into increasingly complex organisms, Ca(2+) acquired more extensive and varied functions. Here, we compare genes encoding proteins that govern Ca(2+) entry and exit across cells or organelles within organisms of early eukaryotic evolution into fungi, plants, and animals. Recent phylogenomics analyses reveal a complex Ca(2+) signaling machinery in the apusozoan protist Thecamonas trahens, a putative unicellular progenitor of Opisthokonta. We compare T. trahens Ca(2+) signaling to that in a marine bikont protist, Aurantiochytrium limacinum, and demonstrate the conservation of key Ca(2+) signaling molecules in the basally diverging alga Cyanophora paradoxa. Particularly, our findings reveal the conservation of the CatSper channel complex in Au. limacinum and C. paradoxa, suggesting that the CatSper complex likely originated from an ancestral Ca(2+) signaling machinery at the root of early eukaryotic evolution prior to the unikont/bikont split.

Tripartite motif containing protein 27 negatively regulates CD4 T cells by ubiquitinating and inhibiting the class II PI3K-C2ß.

The K(+) channel KCa3.1 is required for Ca(2+) influx and the subsequent activation of CD4 T cells. The class II phosphatidylinositol 3 kinase C2ß (PI3KC2ß) is activated by the T-cell receptor (TCR) and is critical for KCa3.1 channel activation. Tripartite motif containing protein 27 (TRIM27) is a member of a large family of proteins that function as Really Interesting New Gene (RING) E3 ubiquitin ligases. We now show that TRIM27 functions as an E3 ligase and mediates lysine 48 polyubiquitination of PI3KC2ß, leading to a decrease in PI3K enzyme activity. By inhibiting PI3KC2ß, TRIM27 also functions to negatively regulate CD4 T cells by inhibiting KCa3.1 channel activity and TCR-stimulated Ca(2+) influx and cytokine production in Jurkat, primary human CD4 T cells, and Th0, Th1, and Th2 CD4 T cells generated from TRIM27(-/-) mice. These findings provide a unique mechanism for regulating class II PI3Ks, and identify TRIM27 as a previously undescribed negative regulator of CD4 T cells.

CDK1 inhibition reduces osteogenesis in endothelial cells in vascular calcification.

Vascular calcification is a severe complication of cardiovascular diseases. Previous studies demonstrated that endothelial lineage cells transitioned into osteoblast-like cells and contributed to vascular calcification. Here, we found that inhibition of cyclin-dependent kinase (CDK) prevented endothelial lineage cells from transitioning to osteoblast-like cells and reduced vascular calcification. We identified a robust induction of CDK1 in endothelial cells (ECs) in calcified arteries and showed that EC-specific gene deletion of CDK1 decreased the calcification. We found that limiting CDK1 induced E-twenty-six specific sequence variant 2 (ETV2), which was responsible for blocking endothelial lineage cells from undergoing osteoblast differentiation. We also found that inhibition of CDK1 reduced vascular calcification in a diabetic mouse model. Together, the results highlight the importance of CDK1 suppression and suggest CDK1 inhibition as a potential option for treating vascular calcification.

Inhibition of endothelial histone deacetylase 2 shifts endothelial-mesenchymal transitions in cerebral arteriovenous malformation models.

Cerebral arteriovenous malformations (AVMs) are the most common vascular malformations worldwide and the leading cause of hemorrhagic strokes that may result in crippling neurological deficits. Here, using newly generated mouse models, we uncovered that cerebral endothelial cells (ECs) acquired mesenchymal markers and caused vascular malformations. Interestingly, we found that limiting endothelial histone deacetylase 2 (HDAC2) prevented cerebral ECs from undergoing mesenchymal differentiation and reduced cerebral AVMs. We found that endothelial expression of HDAC2 and enhancer of zeste homolog 1 (EZH1) was altered in cerebral AVMs. These alterations changed the abundance of H4K8ac and H3K27me in the genes regulating endothelial and mesenchymal differentiation, which caused the ECs to acquire mesenchymal characteristics and form AVMs. Together, this investigation demonstrated that the induction of HDAC2 altered specific histone modifications, which resulted in mesenchymal characteristics in the ECs and cerebral AVMs. The results provided insight into the epigenetic impact on AVMs.

Two-step regulation by matrix Gla protein in brown adipose cell differentiation.

OBJECTIVE: Bone morphogenetic protein (BMP) signaling is intricately involved in adipose tissue development. BMP7 together with BMP4 have been implicated in brown adipocyte differentiation but their roles during development remains poorly specified. Matrix Gla protein (MGP) inhibits BMP4 and BMP7 and is expressed in endothelial and progenitor cells. The objective was to determine the role of MGP in brown adipose tissue (BAT) development. METHODS: The approach included global and cell-specific Mgp gene deletion in combination with RNA analysis, immunostaining, thermogenic activity, and in vitro studies. RESULTS: The results revealed that MGP directs brown adipogenesis at two essential steps. Endothelial-derived MGP limits triggering of white adipogenic differentiation in the perivascular region, whereas MGP derived from adipose cells supports the transition of CD142-expressing progenitor cells to brown adipogenic maturity. Both steps were important to optimize the thermogenic function of BAT. Furthermore, MGP derived from both sources impacted vascular growth. Reduction of MGP in either endothelial or adipose cells expanded the endothelial cell population, suggesting that MGP is a factor in overall plasticity of adipose tissue. CONCLUSION: MGP displays a dual and cell-specific function in BAT, essentially creating a "cellular shuttle" that coordinates brown adipogenic differentiation with vascular growth during development.

Ancestral Ca2+ signaling machinery in early animal and fungal evolution.

Animals and fungi diverged from a common unicellular ancestor of Opisthokonta, yet they exhibit significant differences in their components of Ca2+ signaling pathways. Many Ca2+ signaling molecules appear to be either animal-specific or fungal-specific, which is generally believed to result from lineage-specific adaptations to distinct physiological requirements. Here, by analyzing the genomic data from several close relatives of animals and fungi, we demonstrate that many components of animal and fungal Ca2+ signaling machineries are present in the apusozoan protist Thecamonas trahens, which belongs to the putative unicellular sister group to Opisthokonta. We also identify the conserved portion of Ca2+ signaling molecules in early evolution of animals and fungi following their divergence. Furthermore, our results reveal the lineage-specific expansion of Ca2+ channels and transporters in the unicellular ancestors of animals and in basal fungi. These findings provide novel insights into the evolution and regulation of Ca2+ signaling critical for animal and fungal biology.

G protein-coupled receptor kinase-5 attenuates atherosclerosis by regulating receptor tyrosine kinases and 7-transmembrane receptors.

OBJECTIVE: G protein-coupled receptor kinase-5 (GRK5) is a widely expressed Ser/Thr kinase that regulates several atherogenic receptors and may activate or inhibit nuclear factor-κB (NF-κB). This study sought to determine whether and by what mechanisms GRK5 affects atherosclerosis. METHODS AND RESULTS: Grk5(-/-)/Apoe(-/-) mice developed 50% greater aortic atherosclerosis than Apoe(-/-) mice and demonstrated greater proliferation of macrophages and smooth muscle cells (SMCs) in atherosclerotic lesions. In Apoe(-/-) mice, carotid interposition grafts from Grk5(-/-) mice demonstrated greater upregulation of cell adhesion molecules than grafts from wild-type mice and, subsequently, more atherosclerosis. By comparing Grk5(-/-) with wild-type cells, we found that GRK5 desensitized 2 key atherogenic receptor tyrosine kinases: the platelet-derived growth factor receptor-ß in SMCs, by augmenting ubiquitination/degradation; and the colony-stimulating factor-1 receptor (CSF-1R) in macrophages, by reducing CSF-1-induced tyrosyl phosphorylation. GRK5 activity in monocytes also reduced migration promoted by the 7-transmembrane receptor for monocyte chemoattractant protein-1 CC chemokine receptor-2. Whereas GRK5 diminished NF-κB-dependent gene expression in SMCs and endothelial cells, it had no effect on NF-κB activity in macrophages. CONCLUSIONS: GRK5 attenuates atherosclerosis through multiple cell type-specific mechanisms, including reduction of SMC and endothelial cell NF-κB activity and desensitization of receptor-specific signaling through the monocyte CC chemokine receptor-2, macrophage CSF-1R, and the SMC platelet-derived growth factor receptor-ß.

Evolutionary genomics reveals the premetazoan origin of opposite gating polarity in animal-type voltage-gated ion channels.

Electrical signaling in animals ensures the rapid and accurate transmission of information, often carried by voltage-gated Na(+), Ca(2+) and K(+) channels that are activated by membrane depolarization. In heart and neurons, a distinct type of ion channel called the hyperpolarization-activated, cyclic nucleotide-regulated (HCN) channel is activated by membrane hyperpolarization. Recent genomic studies have revealed that animal-type voltage-gated Na(+) channels (Liebeskind BJ, et al. 2011. Proc Natl Acad Sci U S A. 108:9154) had evolved in choanoflagellates, one of the unicellular relatives of animals. To date, HCN channels have been considered to be animal-specific. Here, we demonstrate the presence of an HCN channel homolog (SroHCN) in the choanoflagellate protist Salpingoeca rosetta. SroHCN contains highly conserved functional domains and sequence motifs that are correlated with the unique biophysical activities of HCN channels. These findings provide novel genomic insights into the evolution of complex electrical signaling before the emergence of multicellular animals.

Cell Transitions Contribute to Glucocorticoid-Induced Bone Loss.

Glucocorticoid-induced bone loss is a toxic effect of long-term therapy with glucocorticoids resulting in a significant increase in the risk of fracture. Here, we find that glucocorticoids reciprocally convert osteoblast-lineage cells into endothelial-like cells. This is confirmed by lineage tracing showing the induction of endothelial markers in osteoblast-lineage cells following glucocorticoid treatment. Functional studies show that osteoblast-lineage cells isolated from glucocorticoid-treated mice lose their capacity for bone formation but simultaneously improve vascular repair. We find that the glucocorticoid receptor directly targets Foxc2 and Osterix, and the modulations of Foxc2 and Osterix drive the transition of osteoblast-lineage cells to endothelial-like cells. Together, the results suggest that glucocorticoids suppress osteogenic capacity and cause bone loss at least in part through previously unrecognized osteoblast-endothelial transitions.

Aurora Kinase A Regulates Cell Transitions in Glucocorticoid-Induced Bone Loss.

Glucocorticoid-induced bone loss is a severe and toxic effect of long-term therapy with glucocorticoids, which are currently prescribed for millions of people worldwide. Previous studies have uncovered that glucocorticoids reciprocally converted osteoblast lineage cells into endothelial-like cells to cause bone loss and showed that the modulations of Foxc2 and Osterix were the causative factors that drove this harmful transition of osteoblast lineage cells. Here, we find that the inhibition of aurora kinase A halts this transition and prevents glucocorticoid-induced bone loss. We find that aurora A interacts with the glucocorticoid receptor and show that this interaction is required for glucocorticoids to modulate Foxc2 and Osterix. Together, we identify a new potential approach to counteracting unwanted transitions of osteoblast lineage cells in glucocorticoid treatment and may provide a novel strategy for ameliorating glucocorticoid-induced bone loss.