Neuropilin 2 in osteoblasts regulates trabecular bone mass in male mice.

Introduction: Neuropilin 2 (NRP2) mediates the effects of class 3 semaphorins and vascular endothelial growth factor and is implicated in axonal guidance and angiogenesis. Moreover, NRP2 expression is suggested to be involved in the regulation of bone homeostasis. Indeed, osteoblasts and osteoclasts express NRP2 and male and female global Nrp2 knockout mice have a reduced bone mass accompanied by reduced osteoblast and increased osteoclast counts. Methods: We first examined the in vitro effect of the calciotropic hormone 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on Nrp2 transcription in osteoblasts. We next generated mice with a conditional deletion of Nrp2 in the osteoblast cell lineage under control of the paired related homeobox 1 promoter and mice with a conditional Nrp2 knockdown in osteoclasts under control of the Lysozyme promoter. Mice were examined under basal conditions or after treatment with either the bone anabolic vitamin D3 analog WY 1048 or with 1,25(OH)2D3. Results and discussion: We show that Nrp2 expression is induced by 1,25(OH)2D3 in osteoblasts and is associated with enrichment of the vitamin D receptor in an intronic region of the Nrp2 gene. In male mice, conditional deletion of Nrp2 in osteoblast precursors and mature osteoblasts recapitulated the bone phenotype of global Nrp2 knockout mice, with a reduced cortical cross-sectional tissue area and lower trabecular bone content. However, female mice with reduced osteoblastic Nrp2 expression display a reduced cross-sectional tissue area but have a normal trabecular bone mass. Treatment with the vitamin D3 analog WY 1048 (0.4 µg/kg/d, 14 days, ip) resulted in a similar increase in bone mass in both genotypes and genders. Deleting Nrp2 from the osteoclast lineage did not result in a bone phenotype, even though in vitro osteoclastogenesis of hematopoietic cells derived from mutant mice was significantly increased. Moreover, treatment with a high dose of 1,25(OH)2D3 (0.5 µg/kg/d, 6 days, ip), to induce osteoclast-mediated bone resorption, resulted in a similar reduction in trabecular and cortical bone mass. In conclusion, osteoblastic Nrp2 expression is suggested to regulate bone homeostasis in a sex-specific manner.

Molecular insights into mineralotropic hormone inter-regulation.

The regulation of mineral homeostasis involves the three mineralotropic hormones PTH, FGF23 and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). Early research efforts focused on PTH and 1,25(OH)2D3 and more recently on FGF23 have revealed that each of these hormones regulates the expression of the other two. Despite early suggestions of transcriptional processes, it has been only recently that research effort have begun to delineate the genomic mechanisms underpinning this regulation for 1,25(OH)2D3 and FGF23; the regulation of PTH by 1,25(OH)2D3, however, remains obscure. We review here our molecular understanding of how PTH induces Cyp27b1 expression, the gene encoding the enzyme responsible for the synthesis of 1,25(OH)2D3. FGF23 and 1,25(OH)2D3, on the other hand, function by suppressing production of 1,25(OH)2D3. PTH stimulates the PKA-induced recruitment of CREB and its coactivator CBP at CREB occupied sites within the kidney-specific regulatory regions of Cyp27b1. PKA activation also promotes the nuclear translocation of SIK bound coactivators such as CRTC2, where it similarly interacts with CREB occupied Cyp27b1 sites. The negative actions of both FGF23 and 1,25(OH)2D3 appear to suppress Cyp27b1 expression by opposing the recruitment of CREB coactivators at this gene. Reciprocal gene actions are seen at Cyp24a1, the gene encoding the enzyme that degrades 1,25(OH)2D3, thereby contributing to the overall regulation of blood levels of 1,25(OH)2D3. Relative to PTH regulation, we summarize what is known of how 1,25(OH)2D3 regulates PTH suppression. These studies suggest that it is not 1,25(OH)2D3 that controls PTH levels in healthy subjects, but rather calcium itself. Finally, we describe current progress using an in vivo approach that furthers our understanding of the regulation of Fgf23 expression by PTH and 1,25(OH)2D3 and provide the first evidence that P may act to induce Fgf23 expression via a complex transcriptional mechanism in bone. It is clear, however, that additional advances will need to be made to further our understanding of the inter-regulation of each of these hormonal genes.

Vitamin D receptor cross-talk with p63 signaling promotes epidermal cell fate.

The vitamin D receptor with its ligand 1,25 dihydroxy vitamin D3 (1,25D3) regulates epidermal stem cell fate, such that VDR removal from Krt14 expressing keratinocytes delays re-epithelialization of epidermis after wound injury in mice. In this study we deleted Vdr from Lrig1 expressing stem cells in the isthmus of the hair follicle then used lineage tracing to evaluate the impact on re-epithelialization following injury. We showed that Vdr deletion from these cells prevents their migration to and regeneration of the interfollicular epidermis without impairing their ability to repopulate the sebaceous gland. To pursue the molecular basis for these effects of VDR, we performed genome wide transcriptional analysis of keratinocytes from Vdr cKO and control littermate mice. Ingenuity Pathway analysis (IPA) pointed us to the TP53 family including p63 as a partner with VDR, a transcriptional factor that is essential for proliferation and differentiation of epidermal keratinocytes. Epigenetic studies on epidermal keratinocytes derived from interfollicular epidermis showed that VDR is colocalized with p63 within the specific regulatory region of MED1 containing super-enhancers of epidermal fate driven transcription factor genes such as Fos and Jun. Gene ontology analysis further implicated that Vdr and p63 associated genomic regions regulate genes involving stem cell fate and epidermal differentiation. To demonstrate the functional interaction between VDR and p63, we evaluated the response to 1,25(OH)2D3 of keratinocytes lacking p63 and noted a reduction in epidermal cell fate determining transcription factors such as Fos, Jun. We conclude that VDR is required for the epidermal stem cell fate orientation towards interfollicular epidermis. We propose that this role of VDR involves cross-talk with the epidermal master regulator p63 through super-enhancer mediated epigenetic dynamics.

Genome-wide analyses of gene expression profile identify key genes and pathways involved in skeletal response to phosphate and 1,25-dihydroxyvitamin D3 in vivo.

Phosphate (P) is an essential element involved in various biological actions, such as bone integrity, energy production, cell signaling and molecular component. P homeostasis is modulated by 4 main tissues; intestine, kidney, bone, and parathyroid gland, where 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), parathyroid hormone and fibroblast growth factor 23 (FGF23) are produced and/or have an influence. In bone, serum P level modulates the production of FGF23 which then controls not only P excretion but also vitamin D metabolism in kidney in an endocrine manner. The hormonally active form of vitamin D, 1,25(OH)2D3, also has a significant effect on skeletal cells via its receptor, the vitamin D receptor, to control gene expression which mediates bone metabolism as well as mineral homeostasis. In this study, we adopted RNA-seq analysis to understand genome-wide skeletal gene expression regulation in response to P and 1,25(OH)2D3. We examined lumbar 5 vertebrae from the mice that were fed P deficient diet for a week followed by an acute high P diet for 3, 6, and 24 h as well as mice treated with 1,25(OH)2D3 intraperitoneally for 6 h. Further identification and exploration of the genes regulated by P and 1,25(OH)2D3 showed that P dynamically modulates the expression of skeletal genes involved in various biological processes while 1,25(OH)2D3 regulates genes highly related to bone metabolism. Our in vivo data were then compared with in vitro data that we previously obtained, which suggests that the gene expression profiles presented in this report mainly represent those of osteocytes. Interestingly, it was found that even though the skeletal response to P is distinguished from that to 1,25(OH)2D3, both factors have an effect on Wnt signaling pathway to modulate bone homeostasis. Taken together, this report presents genome-wide data that provide a foundation to understand molecular mechanisms by which skeletal cells respond to P and 1,25(OH)2D3.

A parathyroid hormone/salt-inducible kinase signaling axis controls renal vitamin D activation and organismal calcium homeostasis.

The renal actions of parathyroid hormone (PTH) promote 1,25-vitamin D generation; however, the signaling mechanisms that control PTH-dependent vitamin D activation remain unknown. Here, we demonstrated that salt-inducible kinases (SIKs) orchestrated renal 1,25-vitamin D production downstream of PTH signaling. PTH inhibited SIK cellular activity by cAMP-dependent PKA phosphorylation. Whole-tissue and single-cell transcriptomics demonstrated that both PTH and pharmacologic SIK inhibitors regulated a vitamin D gene module in the proximal tubule. SIK inhibitors increased 1,25-vitamin D production and renal Cyp27b1 mRNA expression in mice and in human embryonic stem cell-derived kidney organoids. Global- and kidney-specific Sik2/Sik3 mutant mice showed Cyp27b1 upregulation, elevated serum 1,25-vitamin D, and PTH-independent hypercalcemia. The SIK substrate CRTC2 showed PTH and SIK inhibitor-inducible binding to key Cyp27b1 regulatory enhancers in the kidney, which were also required for SIK inhibitors to increase Cyp27b1 in vivo. Finally, in a podocyte injury model of chronic kidney disease-mineral bone disorder (CKD-MBD), SIK inhibitor treatment stimulated renal Cyp27b1 expression and 1,25-vitamin D production. Together, these results demonstrated a PTH/SIK/CRTC signaling axis in the kidney that controls Cyp27b1 expression and 1,25-vitamin D synthesis. These findings indicate that SIK inhibitors might be helpful for stimulation of 1,25-vitamin D production in CKD-MBD.

Genomic mechanisms controlling renal vitamin D metabolism.

Vitamin D metabolism centers on regulation in the kidney of CYP27B1 induction by PTH, suppression by FGF23 and 1,25(OH)2D3, and reciprocal CYP24A1 suppression by PTH, and induction by FGF23 and 1,25(OH)2D3. This coordinated genomic regulation through enhancer modules results in the production and dynamic maintenance of circulating endocrine 1,25(OH)2D3 which, together with PTH and FGF23, controls mineral homeostasis. We discovered enhancers near Cyp27b1 in the mouse kidney located within intronic regions of Mettl1 and Mettl21b genes. These kidney-specific enhancers ("M1", "M21") control Cyp27b1. Through CRISPR/Cas deletion, we found that PTH activation of Cyp27b1 is lost with deletion of M1, whereas FGF23 suppression is lost with deletion of M21. The combination of both deletions (M1/M21-DIKO) eliminated the suppression by 1,25(OH)2D3. Cyp24a1 activation by 1,25(OH)2D3 is controlled by a promoter proximal pair of VDREs as well as a distal region - 35 to - 37 kb (DS2). We also found that FGF23 activation and PTH suppression of Cyp24a1 was located in a region - 21 to - 37 kb downstream (DS1). More recently, using in vivo ChIP-seq in mouse kidney, we demonstrate that PTH activation rapidly induces increased recruitment of pCREB and its coactivators, CBP and CRTC2, to the M1 and M21 enhancers near the Cyp27b1 gene. At distal enhancers of the Cyp24a1 gene, PTH suppression promotes dismisses CBP with only minor changes in pCREB and CRTC2 occupancy, all of which correlate with a suppression of basal histone acetylation across this locus and reduced transcripts. Surprisingly, we find that 1,25(OH)2D3 suppression increases the occupancy of CRTC2 in the M1 enhancer, a novel observation for CRTC2 and/or 1,25(OH)2D3 action. The suppressive actions of 1,25(OH)2D3 and FGF23 at the Cyp27b1 gene are associated with a reduction in CBP recruitment at these enhancers. Although FGF23-regulated transcription factors remain unknown, we hypothesize that VDR occupancy induced at the M1 and M21 enhancers by 1,25(OH)2D3 likely disrupts or competes with the active conformation of these CREB modules thereby preventing full induction by PTH. Our findings show coactivators such as CRTC2 and CBP contribute to Cyp27b1 and Cyp24a1 transcription and provide molecular insight into the coordinated mechanistic actions of PTH, FGF23, and 1,25(OH)2D3 in the kidney that regulate mineral homeostasis.

Impact of age on shoulder range of motion and strength.

Background: Total shoulder arthroplasty (TSA) is a surgical technique commonly used to treat patients with arthritis and rotator cuff deficiency. Its purpose is to reduce pain and improve shoulder function, namely range of motion (ROM) and strength. While shoulder ROM and strength have been studied extensively in patients with various shoulder pathologies, there is a dearth of knowledge with regard to the asymptomatic population. Methods: A cross-sectional study was conducted in the outpatient orthopaedic clinic following institutional review board approval. Patients 18 years of age and older with at least one asymptomatic and healthy shoulder with no prior history of shoulder surgery, injury, or pain were enrolled in the study. Demographic information, ROM, and strength measurements were collected for 256 shoulders, evenly stratified into groups by age and sex. A goniometer was used to measure forward elevation, abduction, and external rotation, and a handheld dynamometer was utilized for measuring strength. Statistical evaluation was conducted using Pearson correlations, analysis of variance, and Bonferroni and Mann-Whitney post hoc tests, with P < .01 indicating a significant difference. Results: Abduction strength (P < .001), external rotation strength (P < .001), and internal rotation strength (P < .001) were negatively correlated with age when viewing the data as a whole and after stratification of males and females. Age and shoulder ROM, namely abduction (P < .001) and forward elevation (P < .001), were also significantly negatively correlated, although internal rotation decreased with age as well. When comparing across age groups, abduction (P = .001) and forward elevation (P = .001) were significantly higher in group 1 (18-35) when compared to group 4 (66+), but external rotation was not significantly different between these groups. External rotation (P = .001) was only significantly different between groups 2 (36-50) and 4. Variation in external rotation strength was also found. Group 4 was found to have significantly less strength than all 3 of the other groups. Conclusion: Shoulder strength significantly decreased with age, with abduction strength and external rotation strength displaying the strongest negative correlations. Decreases in strength were most prominent in patients 66 years of age and above. Shoulder ROM was not as tightly correlated with age, although abduction, forward elevation, and internal rotation were found to generally decrease over time. Differences in external rotation were not clinically significant. These correlations provide useful controls for patients of various ages regarding their clinical outcomes when presenting with shoulder pathology. Variations in current literature allow this study to verify the impact of age on shoulder ROM and strength.

Rapid genomic changes by mineralotropic hormones and kinase SIK inhibition drive coordinated renal Cyp27b1 and Cyp24a1 expression via CREB modules.

Vitamin D metabolism centers on kidney regulation of Cyp27b1 by mineralotropic hormones, including induction by parathyroid hormone (PTH), suppression by fibroblast growth factor 23 (FGF23) and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), and reciprocal regulations for Cyp24a1. This coordinated genomic regulation results in production of endocrine 1,25(OH)2D3, which, together with PTH and FGF23, controls mineral homeostasis. However, how these events are coordinated is unclear. Here, using in vivo chromatin immunoprecipitation sequencing in mouse kidney, we demonstrate that PTH activation rapidly induces increased recruitment of phosphorylated (p-133) CREB (pCREB) and its coactivators, CBP (CREB-binding protein) and CRTC2 (CREB-regulated transcription coactivator 2), to previously defined kidney-specific M1 and M21 enhancers near the Cyp27b1 gene. At distal enhancers of the Cyp24a1 gene, PTH suppression dismisses CBP with only minor changes in pCREB and CRTC2 occupancy, all of which correlate with decreased genomic activity and reduced transcripts. Treatment of mice with salt-inducible kinase inhibitors (YKL-05-099 and SK-124) yields rapid genomic recruitment of CRTC2 to Cyp27b1, limited interaction of CBP, and a transcriptional response for both Cyp27b1 and Cyp24a1 that mirrors the actions of PTH. Surprisingly, we find that 1,25(OH)2D3 suppression increases the occupancy of CRTC2 in the M1 enhancer, a novel observation for CRTC2 and 1,25(OH)2D3 action. Suppressive actions of 1,25(OH)2D3 and FGF23 at the Cyp27b1 gene are associated with reduced CBP recruitment at these CREB-module enhancers that disrupts full PTH induction. Our findings show that CRTC2 contributes to transcription of both Cyp27b1 and Cyp24a1, demonstrate salt-inducible kinase inhibition as a key modulator of vitamin D metabolism, and provide molecular insight into the coordinated mechanistic actions of PTH, FGF23, and 1,25(OH)2D3 in the kidney that regulate mineral homeostasis.

Deletion of a putative promoter-proximal Tnfsf11 regulatory region in mice does not alter bone mass or Tnfsf11 expression in vivo.

The cytokine RANKL is essential for osteoclast formation during physiological and pathological bone resorption. RANKL also contributes to lymphocyte production, development of lymph nodes and mammary glands, as well as other biological activities. Transcriptional control of the Tnfsf11 gene, which encodes RANKL, is complex and involves distant regulatory regions. Nevertheless, cell culture studies suggest that an enhancer region near the transcription start site is involved in the control of Tnfsf11 expression by hormones such as 1,25-(OH)2 vitamin D3 and parathyroid hormone, as well as the sympathetic nervous system. To address the significance of this region in vivo, we deleted the sequence between -510 to -1413 bp, relative to Tnfsf11 exon 1, from mice using CRISPR-based gene editing. MicroCT analysis of the femur and fourth lumbar vertebra of enhancer knockout mice showed no differences in bone mass compared to wild type littermates at 5 weeks and 6 months of age, suggesting no changes in osteoclast formation. RNA extracted from the tibia, fifth lumbar vertebra, thymus, and spleen at 6 months of age also showed no reduction in Tnfsf11 mRNA abundance between these groups. However, maximal stimulation of Tnfsf11 mRNA abundance in cultured stromal cells by PTH was reduced approximately 40% by enhancer deletion, while stimulation by 1,25-(OH)2 vitamin D3 was unaffected. The abundance of B and T lymphocytes in the bone marrow did not differ between genotypes. These results demonstrate that the region between -510 and -1413 does not contribute to Tnfsf11 expression, osteoclast support, or lymphocyte production in mice under normal physiological conditions but may be involved in situations of elevated parathyroid hormone.

Genomic Mechanisms Governing Mineral Homeostasis and the Regulation and Maintenance of Vitamin D Metabolism.

Our recent genomic studies identified a complex kidney-specific enhancer module located within the introns of adjacent Mettl1 (M1) and Mettl21b (M21) genes that mediate basal and PTH induction of Cyp27b1, as well as suppression by FGF23 and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. The tissue specificity for this regulatory module appears to be localized exclusively to renal proximal tubules. Gross deletion of these segments in mice has severe consequences on skeletal health, and directly affects Cyp27b1 expression in the kidney. Deletion of both the M1 and M21 submodules together almost completely eliminates basal Cyp27b1 expression in the kidney, creating a renal specific pseudo-null mouse, resulting in a systemic and skeletal phenotype similar to that of the Cyp27b1-KO mouse caused by high levels of both 25-hydroxyvitamin D3 [25(OH)D3] and PTH and depletion of 1,25(OH)2D3. Cyp24a1 levels in the double KO mouse also decrease because of compensatory downregulation of the gene by elevated PTH and reduced FGF23 that is mediated by an intergenic module located downstream of the Cyp24a1 gene. Outside of the kidney in nonrenal target cells (NRTCs), expression of Cyp27b1 in these mutant mice was unaffected. Dietary normalization of calcium, phosphate, PTH, and FGF23 rescues the aberrant phenotype of this mouse and normalizes the skeleton. In addition, both the high levels of 25(OH)D3 were reduced and the low levels of 1,25(OH)2D3 were fully eliminated in these mutant mice as a result of the rescue-induced normalization of renal Cyp24a1. Thus, these hormone-regulated enhancers for both Cyp27b1 and Cyp24a1 in the kidney are responsible for the circulating levels of 1,25(OH)2D3 in the blood. The retention of Cyp27b1 and Cyp24a1 expression in NRTCs of these endocrine 1,25(OH)2D3-deficient mice suggests that this Cyp27b1 pseudo-null mouse will provide a model for the future exploration of the role of NRTC-produced 1,25(OH)2D3 in the hormone's diverse noncalcemic actions in both health and disease. © 2020 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Single incision latissimus dorsi surgical technique: a three button repair.

Due to the infrequent occurrence of latissimus dorsi insertional avulsions or tendon ruptures, there is no clear evidence on the optimal surgical fixation strategy. A three suture unicortical button repair technique through a single incision offers an anatomic reconstruction of the broad insertional footprint with adequate exposure. This fixation strategy is the preferred technique by the senior author.

Deletion of Mediator 1 suppresses TGFß signaling leading to changes in epidermal lineages and regeneration.

Epidermal lineages and injury induced regeneration are controlled by transcriptional programs coordinating cellular signaling and epigenetic regulators, but the mechanism remains unclear. Previous studies showed that conditional deletion of the transcriptional coactivator Mediator 1 (Med1) changes epidermal lineages and accelerates wound re-epithelialization. Here, we studied a molecular mechanism by which Med1 facilitates these processes, in particular, by focusing on TGFß signaling through genome wide transcriptome analysis. The expression of the TGF ligands (Tgfß1/ß2) and their downstream target genes is decreased in both normal and wounded Med1 null skin. Med1 silencing in cultured keratinocytes likewise reduces the expression of the ligands (TGFß1/ß2) and diminishes activity of TGFß signaling as shown by decreased p-Smad2/3. Silencing Med1 increases keratinocyte proliferation and migration in vitro. Epigenetic studies using chromatin immuno-precipitation and next generation DNA sequencing reveals that Med1 regulates transcription of TGFß components by forming large clusters of enhancers called super-enhancers at the regulatory regions of the TGFß ligand and SMAD3 genes. These results demonstrate that Med1 is required for the maintenance of the TGFß signaling pathway. Finally, we show that pharmacological inhibition of TGFß signaling enhances epidermal lineages and accelerates wound re-epithelialization in skin similar to that seen in the Med1 null mice, providing new insights into epidermal regeneration.

Mechanistic homeostasis of vitamin D metabolism in the kidney through reciprocal modulation of Cyp27b1 and Cyp24a1 expression.

Cyp27b1 and Cyp24a1 are reciprocally regulated in the kidney by the key hormones PTH, FGF23, and 1,25(OH)2D3. Our recent genomic studies in mice identified a complex kidney-specific enhancer module located within the introns of adjacent Mettl1 (M1) and Mettl21b (M21) genes that mediate basal and PTH induction of Cyp27b1 as well as suppression by FGF23 and 1,25(OH)2D3. Gross deletion of these segments in mice has severe consequences on skeletal health, and directly affects Cyp27b1 expression in the kidney. Deletion of both M1 and M21 submodules together fully eliminates basal Cyp27b1 expression in the kidney, leading to a systemic and skeletal phenotype similar to that of the Cyp27b1-KO mouse due to depletion of 1,25(OH)2D3 and high PTH. Cyp24a1 levels in the double KO mouse were low due to compensatory regulation by elevated PTH and reduced FGF23. However, expression of Cyp27b1 and retention of its regulation by inflammation (LPS) in the NRTCs remained unperturbed. Dietary normalization of calcium, phosphate, PTH, and FGF23 rescues this aberrant phenotype and normalizes the skeletal issues. Cyp24a1 is controlled by its own unique enhancers for 1,25(OH)2D3, FGF23, and PTH. We were also able to eliminate these activities in mice. Collectively, the hormone-mediated enhancer regulation of both Cyp27b1 and Cyp24a1 in the kidney is responsible for the circulating levels of 1,25(OH)2D3 in the blood which in turn primarily affects calcium and phosphate regulation. Importantly, we can now manipulate this system with our enhancer deletion animal models to study 1,25(OH)2D3 production in non-renal target cells and tissues not only in disease, where it is known to affect the immune system, but also in healthy individuals. Here we will review our studies that have defined a finely balanced homeostatic control mechanism employed by PTH and FGF23 with catastrophic toxicity protection from 1,25(OH)2D3 in the genomic regulation of vitamin D metabolism and its accompanied control of mineral maintenance.

Transcutaneous electrical nerve stimulation improves fatigue performance of the treated and contralateral knee extensors.

PURPOSE: Transcutaneous electrical nerve stimulation (TENS) can reduce acute and chronic pain. Unilateral fatigue can produce discomfort in the affected limb and force and activation deficits in contralateral non-exercised muscles. TENS-induced local pain analgesia effects on non-local fatigue performance are unknown. Hence, the aim of the study was to determine if TENS-induced pain suppression would augment force output during a fatiguing protocol in the treated and contralateral muscles. METHODS: Three experiments were integrated for this article. Following pre-tests, each experiment involved 20 min of TENS, sham, or a control condition on the dominant quadriceps. Then either the TENS-treated quadriceps (TENS_Treated) or the contralateral quadriceps (TENS_Contra) was tested. In a third experiment, the TENS and sham conditions involved two\; 100-s isometric maximal voluntary contractions (MVC) (30-s recovery) followed by testing of the contralateral quadriceps (TENS_Contra-Fatigue). Testing involved single knee extensors (KE) MVCs (pre- and post-test) and a post-test 30% MVC to task failure. RESULTS: The TENS-treated study induced greater (p = 0.03; 11.0%) time to KE (treated leg) failure versus control. The TENS_Contra-Fatigue induced significant (p = 0.04; 11.7%) and near-significant (p = 0.1; 7.1%) greater time to contralateral KE failure versus sham and control, respectively. There was a 14.5% (p = 0.02) higher fatigue index with the TENS (36.2 ± 10.1%) versus sham (31.6 ± 10.6%) conditions in the second fatigue intervention set (treated leg). There was no significant post-fatigue KE fatigue interaction with the TENS_Contra. CONCLUSIONS: Unilateral TENS application to the dominant KE prolonged time to failure in the treated and contralateral KE suggesting a global pain modulatory response.

A Control Region Near the Fibroblast Growth Factor 23 Gene Mediates Response to Phosphate, 1,25(OH)2D3, and LPS In Vivo.

Fibroblast growth factor 23 (FGF23) is a bone-derived hormone involved in the control of phosphate (P) homeostasis and vitamin D metabolism. Despite advances, however, molecular details of this gene's regulation remain uncertain. In this report, we created mouse strains in which four epigenetically marked FGF23 regulatory regions were individually deleted from the mouse genome using CRISPR/Cas9 gene-editing technology, and the consequences of these mutations were then assessed on Fgf23 expression and regulation in vivo. An initial analysis confirmed that bone expression of Fgf23 and circulating intact FGF23 (iFGF23) were strongly influenced by both chronic dietary P treatment and acute injection of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. However, further analysis revealed that bone Fgf23 expression and iFGF23 could be rapidly upregulated by dietary P within 3 and 6 hours, respectively; this acute upregulation was lost in the FGF23-PKO mouse containing an Fgf23 proximal enhancer deletion but not in the additional enhancer-deleted mice. Of note, prolonged dietary P treatment over several days led to normalization of FGF23 levels in the FGF23-PKO mouse, suggesting added complexity associated with P regulation of FGF23. Treatment with 1,25(OH)2D3 also revealed a similar loss of Fgf23 induction and blood iFGF23 levels in this mouse. Finally, normal lipopolysaccharide (LPS) induction of Fgf23 expression was also compromised in the FGF23-PKO mouse, a result that, together with our previous report, indicates that the action of LPS on Fgf23 expression is mediated by both proximal and distal Fgf23 enhancers. These in vivo data provide key functional insight into the genomic enhancers through which Fgf23 expression is mediated.

A chromatin-based mechanism controls differential regulation of the cytochrome P450 gene Cyp24a1 in renal and non-renal tissues.

Cytochrome P450 family 27 subfamily B member 1 (CYP27B1) and CYP24A1 function to maintain physiological levels of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) in the kidney. Renal Cyp27b1 and Cyp24a1 expression levels are transcriptionally regulated in a highly reciprocal manner by parathyroid hormone (PTH), fibroblast growth factor 23 (FGF23), and 1,25(OH)2D3 In contrast, Cyp24a1 regulation in nonrenal target cells (NRTCs) is limited to induction by 1,25(OH)2D3 Herein, we used ChIP-Seq analyses of mouse tissues to identify regulatory regions within the Cyp24a1 gene locus. We found an extended region downstream of Cyp24a1 containing a cluster of sites, termed C24-DS1, binding PTH-sensitive cAMP-responsive element-binding protein (CREB) and a cluster termed C24-DS2 binding the vitamin D receptor (VDR). VDR-occupied sites were present in both the kidney and NRTCs, but pCREB sites were occupied only in the kidney. We deleted each segment in the mouse and observed that although the overt phenotypes of both cluster deletions were unremarkable, RNA analysis in the C24-DS1-deleted strain revealed a loss of basal renal Cyp24a1 expression, total resistance to FGF23 and PTH regulation, and secondary suppression of renal Cyp27b1; 1,25(OH)2D3 induction remained unaffected in all tissues. In contrast, loss of the VDR cluster in the C24-DS2-deleted strain did not affect 1,25(OH)2D3 induction of renal Cyp24a1 expression yet reduced but did not eliminate Cyp24a1 responses in NRTCs. We conclude that a chromatin-based mechanism differentially regulates Cyp24a1 in the kidney and NRTCs and is essential for the specific functions of Cyp24a1 in these two tissue types.

Targeted genomic deletions identify diverse enhancer functions and generate a kidney-specific, endocrine-deficient Cyp27b1 pseudo-null mouse.

Vitamin D3 is terminally bioactivated in the kidney to 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) via cytochrome P450 family 27 subfamily B member 1 (CYP27B1), whose gene is regulated by parathyroid hormone (PTH), fibroblast growth factor 23 (FGF23), and 1,25(OH)2D3 Our recent genomic studies in the mouse have revealed a complex kidney-specific enhancer module within the introns of adjacent methyltransferase-like 1 (Mettl1) and Mettl21b that mediate basal and PTH-induced expression of Cyp27b1 and FGF23- and 1,25(OH)2D3-mediated repression. Gross deletion of these segments in mice has severe effects on Cyp27b1 regulation and skeletal phenotype but does not affect Cyp27b1 expression in nonrenal target cells (NRTCs). Here, we report a bimodal activity in the Mettl1 intronic enhancer with components responsible for PTH-mediated Cyp27b1 induction and 1,25(OH)2D3-mediated repression and additional activities, including FGF23 repression, within the Mettl21b enhancers. Deletion of both submodules eliminated basal Cyp27b1 expression and regulation in the kidney, leading to systemic and skeletal phenotypes similar to those of Cyp27b1-null mice. However, basal expression and lipopolysaccharide-induced regulation of Cyp27b1 in NRTCs was unperturbed. Importantly, dietary normalization of calcium, phosphate, PTH, and FGF23 rescued the skeletal phenotype of this mutant mouse, creating an ideal in vivo model to study nonrenal 1,25(OH)2D3 production in health and disease. Finally, we confirmed a conserved chromatin landscape in human kidney that is similar to that in mouse. These findings define a finely balanced homeostatic mechanism involving PTH and FGF23 together with protection from 1,25(OH)2D3 toxicity that is responsible for both adaptive vitamin D metabolism and mineral regulation.

The Phosphorylated Estrogen Receptor α (ER) Cistrome Identifies a Subset of Active Enhancers Enriched for Direct ER-DNA Binding and the Transcription Factor GRHL2.

Posttranslational modifications are key regulators of protein function, providing cues that can alter protein interactions and cellular location. Phosphorylation of estrogen receptor α (ER) at serine 118 (pS118-ER) occurs in response to multiple stimuli and is involved in modulating ER-dependent gene transcription. While the cistrome of ER is well established, surprisingly little is understood about how phosphorylation impacts ER-DNA binding activity. To define the pS118-ER cistrome, chromatin immunoprecipitation sequencing was performed on pS118-ER and ER in MCF-7 cells treated with estrogen. pS118-ER occupied a subset of ER binding sites which were associated with an active enhancer mark, acetylated H3K27. Unlike ER, pS118-ER sites were enriched in GRHL2 DNA binding motifs, and estrogen treatment increased GRHL2 recruitment to sites occupied by pS118-ER. Additionally, pS118-ER occupancy sites showed greater enrichment of full-length estrogen response elements relative to ER sites. In an in vitro DNA binding array of genomic binding sites, pS118-ER was more commonly associated with direct DNA binding events than indirect binding events. These results indicate that phosphorylation of ER at serine 118 promotes direct DNA binding at active enhancers and is a distinguishing mark for associated transcription factor complexes on chromatin.