Control of calcium metabolism Calcium and phosphorus homeostasis Primary elements of blood tissue are calcium (Ca) and phosphorus (P). - up to 65 % of bone weight - almost all Ca and P supply, half of supply of Mg in human body - Essential role of these elements in physiological processes Bone tissue - 99 % of overall Ca, of it 99 % in mineral component - 1 % - quickly mobilizable and convertible (ICF - ECF) Extra- and intracellular calcium ICF ECF MIT ER cca 1mM cca 100 nM cca 100 mMcca 25 mM Ca2+-ATPase Ca2+ IK Na+/Ca2+ exchanger VDAC/ Ca2+-uniporter mNCX Calcium and its intake Stomach - Gastric juice and role of HCl - Signalization connected to HCl production Calcium absorption - 25 – 60 % - Age - Dietary habits and calcium content in diet - Bone tissue requirements - Vitamin D Bone tissue is crucial calcium and phosphorus storage tissue. Age-related negative calcium balance is an osteoporosis risk factor. Small intestine - Duodenum a jejunum – 90 % - Adaptive intake – duodenum and ileum Calcium absorption and stomach Mechanisms of calcium absorption Paracellular - Luminal electrochemical gradient - Integrity of intercellular connections - Claudins and their role in paracellular transport Transcellular - TRPV6 and associated proteins - Recyclation of TRPV6 - Alternative mechanisms? VitaminD Glucocorticoids Estradiol Prolactin + +- Calcium on blood (calcemia) GlomerularfiltrationYES Endocrinecontrol GlomerularfiltrationNO Calcium excretion - 98 % of filtered Ca is reabsorbed - 70 % proximal tubule - 20 % thick ascending limb of HL - 5 % collecting duct - 2 % urine - CaSR (TALH) - Paracellin-1 - PTH Bone matrix and bone mineral Collagen type I = most important protein of bone matrix Trombospondin Fibronectin Matrix Gla protein Osteocalcin Biglykan Decorin Bone sialoprotein Osteopontin Osteoadherin Vitamin K-dependent g carboxylation and phosphorylation Ca affinity and mineralization Signaling+haematopoiesis Mineralization = production of small hydroxyapatite crystals (Ca, phosphates, carbonates, Mg, Na, K) External mechanism – alkalic phosphatase Internal mechanism – phospho1 (Phosphoethanolamine/ phosphocholine phosphatase ) Cleavage of pyrophosphate Phosphate availability for mineralization Vesicle formation (matrix) Collagen and its arrangement Ca, P, and AF availability Diet, calcium in diet, calcium/phosphorus in ECF Deposition of calcium SIEBLINGs - Osteopontin, DMP-1 (OC) - Bone sialoprotein, MEPE Endopeptidases, PHEX – FGF23 Bone tissue and its remodeling Bone remodeling cycle. Bone remodeling occurs in four phases: (1) Activation phase: In this phase, MCSF and RANKL promote the differentiation of osteoclast precursors into osteoclasts. (2) Resorption phase: During this phase mature osteoclasts with unique ruffled borders induce resorption of bone by secreting cathepsin K, and H + in the sealing zone. After resorption osteoclasts detach from the surface of the bone and undergo apoptosis. (3) Reversal phase: In the reversal phase osteoblasts' precursors get differentiated into mature osteoblasts and are recruited to the resorption site. (4) Formation phase: In this phase osteoblasts get occupied in the resorbed lacuna and start depositing the bone matrix. After the formation phase, osteoid gets mineralized and bone surface returns to resting phase with bone lining cells. Bhardwaj A, Sapra L, Tiwari A, Mishra P, Sharma S, Srivastava R: “Osteomicrobiology”: The Nexus Between Bone and Bugs. Frontiers in Microbiology 2022, 12. Bone tissue and its remodeling Osteocytes (OC) - Metabolic activity - PTH receptors - Communication with bone surface - Mechanic sensing - RANKL production - Direct degradation of bone tissue (osteocytic osteolysis) - Adaptive remodeling Osteoblasts (OB) - Bone matrix production - Production of collagen and noncollagen peptides + their orientation - Regulation by hormones, local factors and cytokines - Differentiation and further fate – apotosis, osteocytes, lining cells - „recruitment“ of other cells – IGF- 1, IGF-2, TGF-b Lining cells - Stimulation of OB differentiation - OC communication - Differentiation to OB stimulated by PTH Osteoclasts (OK) - Bone tissue reabsorption Bone mechanosensing RANK/RANKL Key factor regulating bone resorption is RANKL/OPG ratio. Osteoclastogenesis (+) RANKL (-) OPG Wnt is synthesized, subjected to Porc-mediated lipidation by palmitoleic acid, and is secreted from cells; Porc is an acyltransferase found in the endoplasmic reticulum. Wls is involved in the extracellular secretion of Wnt. Lipidation by palmitoleic acid is required for the binding of Wnt to Wls. Wls-deficient cells failed to secrete all Wnt ligands. Wnt ligands activate β-catenindependent canonical and -independent non-canonical signals. β-catenin-dependent canonical signal induces bone formation through promotion of osteoblastogenesis and OPG expression. β-catenin-independent non-canonical signals enhance LRP5/6 expression, thereby promoting osteoblast differentiation. OPG: osteoprotegerin, Porc: porcupine, Wls: wntless. RANK/RANKL Wnt signaling pathway Haffner-Luntzer M: Experimental agents to improve fracture healing: utilizing the WNT signaling pathway. Injury 2020, 52. Sclerostin I: Inhibition of proliferation and differentiation of osteoprogenitor/pre-osteoblastic cells, as well as decreased activation of mature osteoblasts; II: decreased mineralization; III: increased apoptosis of the osteogenic cells; IV: maintenance of bone lining cells in their quiescent state; V: regulation of osteocyte maturation and osteocytic osteolysis; VI: stimulation of bone resorption. Rauner M, Taipaleenmäki H, Tsourdi E, Winter L: Osteoporosis Treatment with Anti-Sclerostin Antibodies— Mechanisms of Action and Clinical Application. Journal of Clinical Medicine 2021, 10:787. https://pharmaceutical-journal.com/article/infographics/osteoporosis-moving-beyond-bisphosphonates Bone tissue resorption by osteoclasts Role of compartmentalization in bone resorption - podosomes Resorption and secretion of bone resorption products - transcytosis Essential role of pH for bone tissue resorption Factors affecting bone tissue remodeling Osteoblasts Lining cells Osteoclasts Remodeling of bone tissue Immediate calcium need - homeostasis Ensuring mechanical requirements Trabecularbone Systemic signals Local signalsResorption takes approx. 2 weeks Mineralization and formation approx. 12 weeks In pathophysiologic conditions is disrupted the continuity of bone tissue resorption and formation. Cytokines - IL-1α, IL-1β, TNF-α, TNF-β, proinflammatory IL (7, 15, 17) Cytokines - IL-4, IL-13, IL- 10, IL-18 TGF-α and EGF, FGF21, FGF23 Prostaglandins Prostaglandins PDGF VEGFA, HIF-1a (+/-) IGF-1 (endo-/paracrine) BMPs (OB, autocrine) Calcium sensing receptors (CASRs) Endocrine regulation of bone tissue – PTH,vitamin D, FGF23 Calcium sensing receptors - CaSR - and PTH secretion CaSR – G-protein coupled receptor - Activation of PLC - Inhibition of cAMP production Various distribution in tissues – all tissues participating in calcium homeostasis - Parathyroid glands - Kidneys - Skin - GIT epithelium, enterocytes - G cells of stomach - CNS Clinical aspects - Mutation – inactivation/activation - familial hypocalciuric hypercalcemia (in.) - Familial hypoparathyroidism with hypercalciuria (ac.) - Calcimimetics – inhibition of PTH secretion + amino acids, peptides, Mg Effect of PTH on osteoclasts is indirect. Pulsatile secretion stimulates osteoblasts, chronic continual osteoclasts. PTH and bone tissue physiology + differentiation of osteoblast precursors + secretion of paracrine and autocrine factors (IGF-1) (-) apoptosis (+) osteocytes – release of Ca, osteocytic osteolysis Clinical application – osteoporosis therapy PTHrP Calcitonin Characteristics - C cells of thyroid gland - Family of peptides (amylin, CGRPs, adrenomedulin) - Different distribution in various tissues - Secretion is determined by level of ionized calcium (CaSR) - Stimulation of secretion: - Glucocorticoids - CGRP - Glucagon - Enteroglucagon - Gastrin - Pentagastrin - Pancreozymin - β-sympatomimetics - Inhibition of secretion - somatostatin Function unclear Functions - Bone tissue - Inhibition of osteoclast motility and differentiation - Inhibition of osteoclast secretion - ATPase inhibition - Kidneys - Increased excretion of Ca – inhibition of resorption (Ca2+ ion channels – LS, Na+/Ca2+ - BM) - Skeleton development? - Skeleton protection during pregnancy? Clinical relevance - Osteoporosis therapy - Paget disease therapy - Treatment of pain (bones metastases) - ! Increased risk of cancer Calcitonin and α-CGRP Glucocorticoids Sex hormones GH, IGF-1 ANS and bone physiology Bone as endocrine organ insulin Decarboxylated OCN (Glu13-OCN) leptin Osteocalcin Osteocalcin Osteocalcin Vitamin D….hormone?...vitamin? Characteristics - Intake with diet or synthesized (UV) - In blood bound to VDBP and albumin - Very small free fraction 1,25(OH)2D – cca 0,4 % PTH, prolactin, calcitonin, GH (+) T3/T4, metabolic acidosis (-) Ca, phosphates, 1,25(OH)2D, FGF23 (-) Ketoconazole VDBP Estrogens (+) 1a-hydroxylase - Expression in various tissues - Keratinocytes - Placenta - Macrophages Different 1a-hydroxylase expression = local tissue homeostasis Different rate of feedback control Cartilage,small intestine Physiological effects of vitamin D VDR - High affinity to 1,25(OH)2D - Level of circulating 1,25(OH)2D - Heterodimer with RXR – coactivators, corepressors Non-genomic effects - Rapid increase of intracellular Ca concentration - PLC activation - Opening of some Ca ion channels - Required VDR presence Vitamin D and Ca absorption/reabsorption - (+) CBP, AP, Ca2+/Mg2+-ATPase - (+) TRPV6 – absorption (GIT) - (+/-) TRPV5 – reabsorption (kidneys) - Calbindin-9K - 1,25(OH)2D-inducible ATP-dependent Ca2+ pump - Na+/Ca2+ exchanger Parathyroid glands - Gene expression regulation - Cell proliferation regulation - (-) PTH gene transcription Bones and bone tissue - (-) collagen synthesis - (+) osteocalcin synthesis - (+) osteoclasts differentiation – osteoclastogenesis - (+) RANKL - Main function – ensuring the stability of the bone microenvironment for mineralization by the standard intake and availability of Ca and phosphates Muscle tissue - (+) uptake AAs - (+) troponin C - Phospholipids metabolism Vitamin D and bone resorption Tentative scheme of direct actions of 1,25(OH)2D/VDR on bone. Normal levels of 1,25(OH)2D act via the VDR in mature osteoblasts to decrease the ratio of RANKL/OPG and reduce osteoclastic bone resorption. As well, 1,25(OH)2D action via the VDR in mature osteoblasts increases the bone formation rate (BFR). The net result is increased cortical and trabecular bone. Increased levels of 1,25(OH)2D acting via the VDR in less mature osteoblasts may increase RANKL/OPG, stimulate osteoclastic bone resorption, and reduce trabecular bone. The action of high levels of 1,25(OH)2D in mature osteoblasts and osteocytes can increase local and systemic inhibitors of osseous mineralization and decrease mineralization of bone leading to osteomalacia. FGF23 – fibroblast growth factor 23 Clinical relevance: - Autosomal dominant hypophosphatemic rickets (ADHR) - Tumor-induced osteomalacia (TIO) - Klotho mutation - Prediction of chronic kidney failure prognosis Characteristics - New hormone? - Overexpression = hypophosphatemia and decrease of 1a 25(OH)D hydroxylation Functions - maintaining normophosphatemia and regulation of vitamin D metabolism - IncrDecreased expression of IIa, IIb, and IIc (NPT) – phosphate transport - eased expression of 24-hydroxylase – inactive form - Klotho = co-receptor Regulation - Phosphorus availability in diet (-) - Serum phosphorus - 1,25(OH)2D - iron FGF23 – fibroblast growth factor 23 Regulation of FGF23 and its autocrine/paracrine effects on bone formation. In supra physiologic conditions, FGF23 acts directly on FGFR3 in a Klotho-independent manner, thereby inhibiting bone formation. Increased FGF23 suppresses differentiated osteoblast activity and TNAP transcription, which subsequently causes PPi accumulation in the ECM and inhibits matrix mineralization. In physiological conditions, the actions of FGF23 on canonical receptors (FGFRsKlotho complex) also downregulate TNAP, decreasing matrix mineralization. However, the upregulation of osteoblastic markers in these conditions may be caused by the shifting of remodelling balance toward bone formation or direct action of FGF23 via canonical receptors. The symbol “?” and dash lines denote issues of controversy and unknown mechanisms, respectively. This figure was generated with publication licensed by BioRender, Toronto, ON, Canada (Agreement number: VC237SOKSX, 19 November 2021). Abbreviations: BALP, Specific bone Alkaline phosphatase; FGF23, Fibroblast growth factor 23; Pi, Inorganic phosphate; PPi, Pyrophosphate; Runx2, Runt-related transcription factor 2; TNAP, Tissue nonspecific alkaline phosphatase; OC, Osteocalcin.