A delicate balance: the challenges of hypoparathyroidism.

A 38-yr-old woman with chronic non-surgical hypoparathyroidism, managed elsewhere, presented to our practice with symptomatic hypocalcemia. At the age of 17, she began to suffer from muscle cramps, paresthesia, and ongoing diffuse pain. It took years before she was correctly diagnosed with hypoparathyroidism. Her symptoms were severe enough that she required emergency room visits several times a year. After she was properly diagnosed and started on calcium and calcitriol therapy, she continued to experience frequent episodes of severe hypocalcemia. She saw multiple healthcare providers who each introduced a new regimen. In addition, poor communication led to her discontinuing her medications altogether. As a result, her calcium levels remained consistently low, and she lost confidence in her prospect for better health. At the time of her visit to our clinic, she had discontinued calcitriol, was taking a large amount of oral calcium daily all at once, and had hypocalcemia. We addressed her concerns, and the challenges she faces with adherence to her medication regimen. We provided her with detailed information about the disease and the reasoning behind her treatment plan. Treatment was initiated with calcium carbonate 600 mg 3 times daily and calcitriol 0.5 mcg once daily. One week after treatment initiation, her test results showed improvement in her albumin-adjusted calcium, phosphorus, and 24-h urine calcium which were all within target range.

Phosphate-induced activation of VEGFR2 leads to caspase-9-mediated apoptosis of hypertrophic chondrocytes.

Low circulating phosphate (Pi) leads to rickets, characterized by expansion of the hypertrophic chondrocytes (HCs) in the growth plate due to impaired HC apoptosis. Studies in HCs demonstrate that Pi activates the Raf/MEK/ERK1/2 and mitochondrial apoptotic pathways. To determine how Pi activates these pathways, a small-molecule screen was undertaken to identify inhibitors of Pi-induced ERK1/2 phosphorylation in HCs. Vascular endothelial growth factor receptor 2 (VEGFR2) was identified as a target. In vitro studies in HCs demonstrate that VEGFR2 inhibitors block Pi-induced pERK1/2 and caspase-9 cleavage. Like Pi, rhVEGF activates ERK1/2 and caspase-9 in HCs and induces phosphorylation of VEGFR2, confirming that Pi activates this signaling pathway in HCs. Chondrocyte-specific depletion of VEGFR2 leads to an increase in HCs, impaired vascular invasion, and a decrease in HC apoptosis. Thus, these studies define a role for VEGFR2 in transducing Pi signals and mediating its effects on growth plate maturation.

Bone of Contention: Helicobacter pylori and Osteoporosis-Is There an Association?

Helicobacter pylori (H. pylori) infection is a common disease that can cause chronic gastritis, peptic ulcers, and gastric cancer. Nevertheless, due to its ability to elicit a systemic inflammatory response, it has also been related to several extra-gastric manifestations including endocrine disorders, such as autoimmune thyroid diseases, diabetes mellitus, dyslipidemia, and obesity. H. pylori infection has also been linked to osteoporosis, although currently available data are equivocal. This brief review will focus on the possible association between H. pylori infection and osteoporosis, a silent disease characterized by decreased bone mass that can increase the occurrence of fractures, disability, and mortality.

Infliximab in inflammatory bowel disease.

Anti-tumor necrosis factor (TNF) therapy has revolutionized the medical treatment of the inflammatory bowel diseases (IBD), Crohn's disease (CD), and ulcerative colitis. Twenty years ago, infliximab became the first anti-TNF agent approved for IBD. Data from randomized controlled trials, large observational cohort studies, postmarketing registries, and meta-analyses show that infliximab is a very effective treatment for moderate to severe IBD with a good safety profile. Infliximab has been also used to treat pouchitis following an ileal pouch-anal anastomosis (IPAA) after restorative proctocolectomy and to prevent postoperative recurrence following an ileocolonic resection for CD with good results. Nevertheless, up to 30% of patients show no clinical benefit following induction and up to 50% lose response over time. Both these unwanted outcomes can be largely explained by inadequate drug concentrations and frequently by the development of antibodies to infliximab. Loss of response can be managed efficiently and often prevented by applying therapeutic drug monitoring. Recently, the first biosimilars of infliximab have been approved and are utilized in clinical practice with comparable efficacy and safety with the originator. This review will mainly focus on the efficacy of infliximab in IBD.

Distinct molecular pathways mediate Mycn and Myc-regulated miR-17-92 microRNA action in Feingold syndrome mouse models.

Feingold syndrome is a skeletal dysplasia caused by loss-of-function mutations of either MYCN (type 1) or MIR17HG that encodes miR-17-92 microRNAs (type 2). Since miR-17-92 expression is transcriptionally regulated by MYC transcription factors, it has been postulated that Feingold syndrome type 1 and 2 may be caused by a common molecular mechanism. Here we show that Mir17-92 deficiency upregulates TGF-ß signaling, whereas Mycn-deficiency downregulates PI3K signaling in limb mesenchymal cells. Genetic or pharmacological inhibition of TGF-ß signaling efficiently rescues the skeletal defects caused by Mir17-92 deficiency, suggesting that upregulation of TGF-ß signaling is responsible for the skeletal defect of Feingold syndrome type 2. By contrast, the skeletal phenotype of Mycn-deficiency is partially rescued by Pten heterozygosity, but not by TGF-ß inhibition. These results strongly suggest that despite the phenotypical similarity, distinct molecular mechanisms underlie the pathoetiology for Feingold syndrome type 1 and 2.

Raf Kinases Are Essential for Phosphate Induction of ERK1/2 Phosphorylation in Hypertrophic Chondrocytes and Normal Endochondral Bone Development.

Hypophosphatemia causes rickets by impairing hypertrophic chondrocyte apoptosis. Phosphate induction of MEK1/2-ERK1/2 phosphorylation in hypertrophic chondrocytes is required for phosphate-mediated apoptosis and growth plate maturation. MEK1/2 can be activated by numerous molecules including Raf isoforms. A- and B-Raf ablation in chondrocytes does not alter skeletal development, whereas ablation of C-Raf decreases hypertrophic chondrocyte apoptosis and impairs vascularization of the growth plate. However, ablation of C-Raf does not impair phosphate-induced ERK1/2 phosphorylation in vitro, but leads to rickets by decreasing VEGF protein stability. To determine whether Raf isoforms are required for phosphate-induced hypertrophic chondrocyte apoptosis, mice lacking all three Raf isoforms in chondrocytes were generated. Raf deletion caused neonatal death and a significant expansion of the hypertrophic chondrocyte layer of the growth plate, accompanied by decreased cleaved caspase-9. This was associated with decreased phospho-ERK1/2 immunoreactivity in the hypertrophic chondrocyte layer and impaired vascular invasion. These data further demonstrated that Raf kinases are required for phosphate-induced ERK1/2 phosphorylation in cultured hypertrophic chondrocytes and perform essential, but partially redundant roles in growth plate maturation.

Ras signaling regulates osteoprogenitor cell proliferation and bone formation.

During endochondral bone development, osteoblasts are continuously differentiated from locally residing progenitor cells. However, the regulation of such endogenous osteoprogenitor cells is still poorly understood mainly due to the difficulty in identifying such cells in vivo. In this paper, we genetically labeled different cell populations of the osteoblast linage using stage-specific, tamoxifen-inducible Cre transgenic mice to investigate their responses to a proliferative stimulus. We have found that overactivation of Kras signaling in type II collagen-positive, immature osteoprogenitor cells, but not in mature osteoblasts, substantially increases the number of their descendant stromal cells and mature osteoblasts, and subsequently increases bone mass. This effect was mediated by both, the extracellular signal-regulated kinase (ERK) and phosphoinositide 3 kinase (PI3K), pathways. Thus we demonstrate that Ras signaling stimulates proliferation of immature osteoprogenitor cells to increase the number of their osteoblastic descendants in a cell-autonomous fashion.

The Vitamin D Receptor Regulates Tissue Resident Macrophage Response to Injury.

Ligand-dependent actions of the vitamin D receptor (VDR) play a pleiotropic role in the regulation of innate and adaptive immunity. The liganded VDR is required for recruitment of macrophages during the inflammatory phase of cutaneous wound healing. Although the number of macrophages in the granulation tissue 2 days after wounding is markedly reduced in VDR knockout (KO) compared with wild-type mice, VDR ablation does not alter macrophage polarization. Parabiosis studies demonstrate that circulatory chimerism with wild-type mice is unable to rescue the macrophage defect in the wounds of VDR KO mice and reveal that wound macrophages are of local origin, regardless of VDR status. Wound cytokine analyses demonstrated a decrease in macrophage colony-stimulating factor (M-CSF) protein levels in VDR KO mice. Consistent with this, induction of M-CSF gene expression by TGFß and 1,25-dihydroxyvitamin D was impaired in dermal fibroblasts isolated from VDR KO mice. Because M-CSF is important for macrophage self-renewal, studies were performed to evaluate the response of tissue resident macrophages to this cytokine. A decrease in M-CSF induced proliferation and cyclin D1 expression was observed in peritoneal resident macrophages isolated from VDR KO mice, suggesting an intrinsic macrophage abnormality. Consistent with this, wound-healing assays in mice with macrophage-specific VDR ablation demonstrate that a normal wound microenvironment cannot compensate for the absence of the VDR in macrophages and thus confirm a critical role for the macrophage VDR in the inflammatory response to injury.

Polycomb repressive complex 2 regulates skeletal growth by suppressing Wnt and TGF-ß signalling.

Polycomb repressive complex 2 (PRC2) controls maintenance and lineage determination of stem cells by suppressing genes that regulate cellular differentiation and tissue development. However, the role of PRC2 in lineage-committed somatic cells is mostly unknown. Here we show that Eed deficiency in chondrocytes causes severe kyphosis and a growth defect with decreased chondrocyte proliferation, accelerated hypertrophic differentiation and cell death with reduced Hif1a expression. Eed deficiency also causes induction of multiple signalling pathways in chondrocytes. Wnt signalling overactivation is responsible for the accelerated hypertrophic differentiation and kyphosis, whereas the overactivation of TGF-ß signalling is responsible for the reduced proliferation and growth defect. Thus, our study demonstrates that PRC2 has an important regulatory role in lineage-committed tissue cells by suppressing overactivation of multiple signalling pathways.

MicroRNA-140 Provides Robustness to the Regulation of Hypertrophic Chondrocyte Differentiation by the PTHrP-HDAC4 Pathway.

Growth plate chondrocytes go through multiple differentiation steps and eventually become hypertrophic chondrocytes. The parathyroid hormone (PTH)-related peptide (PTHrP) signaling pathway plays a central role in regulation of hypertrophic differentiation, at least in part, through enhancing activity of histone deacetylase 4 (HDAC4), a negative regulator of MEF2 transcription factors that drive hypertrophy. We have previously shown that loss of the chondrocyte-specific microRNA (miRNA), miR-140, alters chondrocyte differentiation including mild acceleration of hypertrophic differentiation. Here, we provide evidence that miR-140 interacts with the PTHrP-HDAC4 pathway to control chondrocyte differentiation. Heterozygosity of PTHrP or HDAC4 substantially impaired animal growth in miR-140 deficiency, whereas these mutations had no effect in the presence of miR-140. miR-140-deficient chondrocytes showed increased MEF2C expression with normal levels of total and phosphorylated HDAC4, indicating that the miR-140 pathway merges with the PTHrP-HDAC4 pathway at the level of MEF2C. miR-140 negatively regulated p38 mitogen-activated protein kinase (MAPK) signaling, and inhibition of p38 MAPK signaling reduced MEF2C expression. These results demonstrate that miR-140 ensures the robustness of the PTHrP/HDAC4 regulatory system by suppressing MEF2C-inducing stimuli. © 2014 American Society for Bone and Mineral Research © 2015 American Society for Bone and Mineral Research.

miRNAs in Bone Development.

Skeletal development is a multistage process during which mesenchymal progenitor cells undergo proliferation and differentiation and subsequently give rise to bone and cartilage forming cells. Each step is regulated by various transcription factors and signaling molecules. microRNAs are small non-coding RNAs that post-transcriptionally regulate gene expression. Several in vivo and in vitro studies have shown that miRNAs play significant roles in skeletal development. Identifying their functions may give insights into the treatment of developmental disorders of the skeleton. This review summarizes miRNAs that have been shown to participate in various stages of skeletal development by targeting crucial factors.

MicroRNAs involved in bone formation.

During skeletal development, mesenchymal progenitor cells undergo a multistage differentiation process in which they proliferate and become bone- and cartilage-forming cells. This process is tightly regulated by multiple levels of regulatory systems. The small non-coding RNAs, microRNAs (miRNAs), post-transcriptionally regulate gene expression. Recent studies have demonstrated that miRNAs play significant roles in all stages of bone formation, suggesting the possibility that miRNAs can be novel therapeutic targets for skeletal diseases. Here, we review the role and mechanism of action of miRNAs in bone formation. We discuss roles of specific miRNAs in major types of bone cells, osteoblasts, chondrocytes, osteoclasts, and their progenitors. Except a few, the current knowledge about miRNAs in bone formation has been obtained mainly by in vitro studies; further validation of these findings in vivo is awaited. We also discuss about several miRNAs of particular interest in the light of future therapies of bone diseases.

MicroRNAs in cartilage development, homeostasis, and disease.

microRNAs (miRNAs) regulate gene expression mainly at the posttranscriptional level. Many different miRNAs are expressed in chondrocytes, and each individual miRNA can regulate hundreds of target genes, creating a complex gene regulatory network. Experimental evidence suggests that miRNAs play significant roles in various aspects of cartilage development, homeostasis, and pathology. The possibility that miRNAs can be novel therapeutic targets for cartilage diseases led to vigorous investigations to understand the role of individual miRNAs in skeletal tissues. Here, we summarize our current understanding of miRNAs in chondrocytes and cartilage. In the first part, we discuss roles of miRNAs in growth plate development and chondrocyte differentiation. In the second part, we put a particular focus on articular cartilage and discuss the significance of variety of findings in the context of osteoarthritis, the most common degenerative joint disease.

Cytokines and insulin resistance after zoledronic acid-induced acute phase response.

Zoledronic acid is known to induce a transient acute phase response (APR). The aim of the study was to investigate whether an APR caused by zoledronic acid administration can induce insulin resistance in post-menopausal osteoporotic women and the potential involvement of different inflammatory markers, cytokines and adipokines to this response. Insulin resistance was calculated by the homeostasis model assessment (HOMA-IR). APR symptoms appeared in 30 post-menopausal osteoporotic women within 24 h and attenuated on day 3 after zoledronic acid infusion. Twenty-eight age- and body mass index-matched, patients without an APR following zoledronic acid administration, served as a control group. In patients with APR, concurrently with a significant increase in serum high sensitive C-reactive protein (hsCRP), interleukin-6 (hsIL-6), tumour necrosis factor-alpha (hsTNF-α) and cortisol levels on days one and two, serum insulin was also significantly elevated, resulting in an increased HOMA-IR. Leptin and resistin significantly increased on day two in contrast to adiponectin which declined, though not statistically significant. The alterations in HOMA-IR were mainly associated to the increase of hsCRP and leptin. In conclusion, zoledronic acid induces an acute, short term insulin resistance, due to an APR, by altering the levels of various adipokines and cytokines.

Autoimmune thyroiditis in benign and malignant thyroid nodules: 16-year results.

BACKGROUND: It is controversial whether autoimmune thyroiditis is associated with higher frequency of papillary thyroid carcinoma (PTC). METHODS: This was a cross-sectional, retrospective study. PTCs were compared to benign nodules regarding the prevalence of autoimmune thyroiditis over 16 years. RESULTS: A similar proportion of autoimmune thyroiditis was observed in both benign and/or malignant nodules. Mean nodule size in cases with autoimmune thyroiditis was smaller than those without autoimmune thyroiditis. Multivariate analysis showed a negative association between the coexistence of autoimmune thyroiditis and lymph node and/or distant metastases. Lymph nodes involvement and distant metastases were lower in the PTC with autoimmune thyroiditis compared to those without autoimmune thyroiditis. Capsular invasion was a strong predictor for distant metastases attenuated by the presence of autoimmune thyroiditis. CONCLUSION: Thyroid nodules with autoimmune thyroiditis are not more likely to be malignant than those without autoimmune thyroiditis. The coexistent autoimmune thyroiditis may be beneficial as a decreased incidence of lymph nodes involvement and distant metastasis was seen in those patients.

let-7 and miR-140 microRNAs coordinately regulate skeletal development.

MicroRNAs (miRNAs) play critical roles in multiple processes of skeletal development. A global reduction of miRNAs in growth plate chondrocytes results in defects in both proliferation and differentiation; however, specific microRNAs responsible for these defects have not been identified. In this study, we provide evidence that let-7 miRNAs and microRNA-140 (miR-140), among other miRNAs expressed in chondrocytes, play major roles in endochondral bone development. We overexpressed lin-28 homolog A (Lin28a) to inhibit let-7 miRNA biogenesis in growth plate chondrocytes. Lin28a overexpression efficiently and specifically reduced let-7 miRNAs and up-regulated let-7 target genes. However, unlike the previous notion that let-7 miRNAs inhibit proliferation and growth, suppression of let-7 miRNAs via Lin28a overexpression decreased proliferation in growth plate chondrocytes, likely through up-regulation of the let-7 target cell cycle regulators cell division cycle 34 (Cdc34) and E2F transcription factor 5 (E2F5). Deficiency of the chondrocyte-specific miRNA, miR-140, causes a differentiation defect in growth plate chondrocytes. Although either Lin28a overexpression or miR-140 deficiency alone caused only mild growth impairment, mice with both miR-140 deficiency and Lin28a overexpression in chondrocytes showed a dramatic growth defect. Deregulation of distinct processes in the absence of these miRNAs synergistically decreased the proliferating chondrocyte mass; miR-140 deficiency reduced differentiation into proliferating chondrocytes, whereas Lin28a overexpression decreased proliferation per se.