Hema Kalyanaraman

Nongenomic Thyroid Hormone Signaling Occurs Through a Plasma Membrane–Localized Receptor

Signaling from a plasma membrane–associated receptor contributes to the effects of thyroid hormones on bones. Rapidly Promoting Bone Growth from the Membrane Thyroid hormones regulate many processes, including bone turnover. By entering cells and binding to nuclear receptors, thyroid hormones induce target gene expression; however, they also stimulate rapid cellular changes that are independent of gene regulation. Kalyanaraman et al. found an alternative form of the thyroid receptor that associated with the cellular plasma membrane of bone cells. Stimulation of this receptor by thyroid hormones increased the numbers of bone cells and protected them from death. Treatment of mice deficient in thyroid hormones with a compound that mimicked signaling from this membrane-associated receptor reversed defects in bone formation, suggesting that this form of thyroid hormone action may be clinically relevant. Thyroid hormone (TH) is essential for vertebrate development and the homeostasis of most adult tissues, including bone. TH stimulates target gene expression through the nuclear thyroid receptors TRα and TRβ; however, TH also has rapid, transcription-independent (nongenomic) effects. We found a previously uncharacterized plasma membrane–bound receptor that was necessary and sufficient for nongenomic TH signaling in several cell types. We determined that this receptor is generated by translation initiation from an internal methionine of TRα, which produces a transcriptionally incompetent protein that is palmitoylated and associates with caveolin-containing plasma membrane domains. TH signaling through this receptor stimulated a pro-proliferative and pro-survival program by increasing the intracellular concentrations of calcium, nitric oxide (NO), and cyclic guanosine monophosphate (cGMP), which led to the sequential activation of protein kinase G II (PKGII), the tyrosine kinase Src, and extracellular signal–regulated kinase (ERK) and Akt signaling. Hypothyroid mice exhibited a cGMP-deficient state with impaired bone formation and increased apoptosis of osteocytes, which was rescued by a direct stimulator of guanylate cyclase. Our results link nongenomic TH signaling to a previously uncharacterized membrane-bound receptor, and identify NO synthase, guanylate cyclase, and PKGII as TH effectors that activate kinase cascades to regulate cell survival and proliferation.

Soluble Guanylate Cyclase as a Novel Treatment Target for Osteoporosis

Osteoporosis is a major health problem leading to fractures that cause substantial morbidity and mortality. Current osteoporosis therapies have significant drawbacks, creating a need for novel bone-anabolic agents. We previously showed that the nitric oxide/cyclic GMP (cGMP)/protein kinase G pathway mediates some of the anabolic effects of estrogens and mechanical stimulation in osteoblasts and osteocytes, leading us to hypothesize that cGMP-elevating agents may have bone-protective effects. We tested cinaciguat, a prototype of a novel class of soluble guanylate cyclase activators, in a mouse model of estrogen deficiency-induced osteoporosis. Compared with sham-operated mice, ovariectomized mice had lower serum cGMP concentrations, which were largely restored to normal by treatment with cinaciguat or low-dose 17β-estradiol. Microcomputed tomography of tibiae showed that cinaciguat significantly improved trabecular bone microarchitecture in ovariectomized animals, with effect sizes similar to those obtained with estrogen replacement therapy. Cinaciguat reversed ovariectomy-induced osteocyte apoptosis as efficiently as estradiol and enhanced bone formation parameters in vivo, consistent with in vitro effects on osteoblast proliferation, differentiation, and survival. Compared with 17β-estradiol, which completely reversed the ovariectomy-induced increase in osteoclast number, cinaciguat had little effect on osteoclasts. Direct guanylate cyclase stimulators have been extremely well tolerated in clinical trials of cardiovascular diseases, and our findings provide proof-of-concept for this new class of drugs as a novel, anabolic treatment strategy for postmenopausal osteoporosis, confirming an important role of nitric oxide/cGMP/protein kinase G signaling in bone.

A Novel, Direct NO Donor Regulates Osteoblast and Osteoclast Functions and Increases Bone Mass in Ovariectomized Mice

Most US Food and Drug Administration (FDA)‐approved treatments for osteoporosis target osteoclastic bone resorption. Only PTH derivatives improve bone formation, but they have drawbacks, and novel bone‐anabolic agents are needed. Nitrates, which generate NO, improved BMD in estrogen‐deficient rats and may improve bone formation markers and BMD in postmenopausal women. However, nitrates are limited by induction of oxidative stress and development of tolerance, and may increase cardiovascular mortality after long‐term use. Here we studied nitrosyl‐cobinamide (NO‐Cbi), a novel, direct NO‐releasing agent, in a mouse model of estrogen deficiency–induced osteoporosis. In murine primary osteoblasts, NO‐Cbi increased intracellular cGMP, Wnt/β‐catenin signaling, proliferation, and osteoblastic gene expression, and protected cells from apoptosis. Correspondingly, in intact and ovariectomized (OVX) female C57Bl/6 mice, NO‐Cbi increased serum cGMP concentrations, bone formation, and osteoblastic gene expression, and in OVX mice, it prevented osteocyte apoptosis. NO‐Cbi reduced osteoclasts in intact mice and prevented the known increase in osteoclasts in OVX mice, partially through a reduction in the RANKL/osteoprotegerin gene expression ratio, which regulates osteoclast differentiation, and partially through direct inhibition of osteoclast differentiation, observed in vitro in the presence of excess RANKL. The positive NO effects in osteoblasts were mediated by cGMP/protein kinase G (PKG), but some of the osteoclast‐inhibitory effects appeared to be cGMP‐independent. NO‐Cbi increased trabecular bone mass in both intact and OVX mice, consistent with its in vitro effects on osteoblasts and osteoclasts. NO‐Cbi is a novel direct NO‐releasing agent that, in contrast to nitrates, does not generate oxygen radicals, and combines anabolic and antiresorptive effects in bone, making it an excellent candidate for treating osteoporosis.

The activity of cGMP-dependent protein kinase Iα is not directly regulated by oxidation-induced disulfide formation at cysteine 43

The type I cGMP-dependent protein kinases (PKGs) are key regulators of smooth muscle tone, cardiac hypertrophy, and other physiological processes. The two isoforms PKGIα and PKGIβ are thought to have unique functions because of their tissue-specific expression, different cGMP affinities, and isoform-specific protein-protein interactions. Recently, a non-canonical pathway of PKGIα activation has been proposed, in which PKGIα is activated in a cGMP-independent fashion via oxidation of Cys43, resulting in disulfide formation within the PKGIα N-terminal dimerization domain. A “redox-dead” knock-in mouse containing a C43S mutation exhibits phenotypes consistent with decreased PKGIα signaling, but the detailed mechanism of oxidation-induced PKGIα activation is unknown. Therefore, we examined oxidation-induced activation of PKGIα, and in contrast to previous findings, we observed that disulfide formation at Cys43 does not directly activate PKGIα in vitro or in intact cells. In transfected cells, phosphorylation of Ras homolog gene family member A (RhoA) and vasodilator-stimulated phosphoprotein was increased in response to 8-CPT-cGMP treatment, but not when disulfide formation in PKGIα was induced by H2O2. Using purified enzymes, we found that the Cys43 oxidation had no effect on basal kinase activity or Km and Vmax values; however, PKGIα containing the C43S mutation was less responsive to cGMP-induced activation. This reduction in cGMP affinity may in part explain the PKGIα loss-of-function phenotype of the C43S knock-in mouse. In conclusion, disulfide formation at Cys43 does not directly activate PKGIα, and the C43S-mutant PKGIα has a higher Ka for cGMP. Our results highlight that mutant enzymes should be carefully biochemically characterized before making in vivo inferences.

Nitric oxide as a mediator of estrogen effects in osteocytes.

Postmenopausal osteoporosis due to estrogen deficiency is a major health problem, and available therapies rely largely on the inhibition of bone resorption, because estrogen replacement is associated with risks. Estrogen promotes bone health in large part by increasing osteocyte survival, but the molecular mechanisms involved are only partly understood. We showed that estradiol stimulates nitric oxide (NO) production in osteocytes, leading to increased cGMP synthesis and activation of cGMP-dependent protein kinases (PKGs). Moreover, we found that 17β-estradiol protects osteocytes against apoptosis via the NO/cGMP signaling pathway: type II PKG mediates estradiol-induced activation of the prosurvival kinases Erk and Akt, whereas type I PKG contributes to prosurvival signaling by directly phosphorylating and inactivating the cell death protein BAD. Preclinical data support an important role of NO in bone biology, and clinical trials suggest that NO donors may prevent bone loss in postmenopausal women. Our data provide novel insights into estrogen signaling through the NO/cGMP/PKG pathway and a rationale for using NO donors and other cGMP-elevating agents for treating postmenopausal osteoporosis.

Protein Kinase G Activation Reverses Oxidative Stress and Restores Osteoblast Function and Bone Formation in Male Mice With Type 1 Diabetes

Bone loss and fractures are underrecognized complications of type 1 diabetes and are primarily due to impaired bone formation by osteoblasts. The mechanisms leading to osteoblast dysfunction in diabetes are incompletely understood, but insulin deficiency, poor glycemic control, and hyperglycemia-induced oxidative stress likely contribute. Here we show that insulin promotes osteoblast proliferation and survival via the nitric oxide (NO)/cyclic guanosine monophosphate (cGMP)/protein kinase G (PKG) signal transduction pathway and that PKG stimulation of Akt provides a positive feedback loop. In osteoblasts exposed to high glucose, NO/cGMP/PKG signaling was reduced due in part to the addition of O-linked N-acetylglucosamine to NO synthase-3, oxidative inhibition of guanylate cyclase activity, and suppression of PKG transcription. Cinaciguat—an NO-independent activator of oxidized guanylate cyclase—increased cGMP synthesis under diabetic conditions and restored proliferation, differentiation, and survival of osteoblasts. Cinaciguat increased trabecular and cortical bone in mice with type 1 diabetes by improving bone formation and osteocyte survival. In bones from diabetic mice and in osteoblasts exposed to high glucose, cinaciguat reduced oxidative stress via PKG-dependent induction of antioxidant genes and downregulation of excess NADPH oxidase-4–dependent H2O2 production. These results suggest that cGMP-elevating agents could be used as an adjunct treatment for diabetes-associated osteoporosis.

Nitric oxide and cyclic GMP functions in bone

Nitric oxide plays a central role in the regulation of skeletal homeostasis. In cells of the osteoblastic lineage, NO is generated in response to mechanical stimulation and estrogen exposure. Via activation of soluble guanylyl cyclase (sGC) and cGMP-dependent protein kinases (PKGs), NO enhances proliferation, differentiation, and survival of bone-forming cells in the osteoblastic lineage. NO also regulates the differentiation and activity of bone-resorbing osteoclasts; here the effects are largely inhibitory and partly cGMP-independent. We review the skeletal phenotypes of mice deficient in NO synthases and PKGs, and the effects of NO and cGMP on bone formation and resorption. We examine the roles of NO and cGMP in bone adaptation to mechanical stimulation. Finally, we discuss preclinical and clinical data showing that NO donors and NO-independent sGC activators may protect against estrogen deficiency-induced bone loss. sGC represents an attractive target for the treatment of osteoporosis.

Targeting NO Signaling for the Treatment of Osteoporosis

Osteoporosis is a major health problem, affecting over 10 million people in the U.S. and leading to fractures associated with significant morbidity and mortality. Normal bone mass is maintained by a balance between the anabolic effects of osteoblasts and catabolic effects of osteoclasts. Most osteoporosis therapies inhibit osteoclast activity; parathyroid hormone is the only FDA-approved agent that increases osteoblast activity, but its efficacy wanes over time, and there is a need for novel bone-anabolic agents. Nitrates, which generate nitric oxide (NO) in vivo, prevent bone loss from estrogen-deficiency in rodents, and some clinical data suggest beneficial effects of nitrates in post-menopausal osteoporosis. Here, we examine the sources of NO and regulation of NO synthesis in bone cells, review the effects of NO in cells of osteoblastic and osteoclastic lineage, and summarize existing preclinical and clinical data to document the skeletal effects of NO in vivo. Based on the anabolic and anti-resorptive effects of NO in bone, novel NO donors and other strategies to enhance NO production and bioavailability in vivo may represent a new treatment strategy for osteoporosis.

NO/GMP as mediators of estrogen effects in bone

Background Osteoporosis is a major health problem, especially in post-menopausal women. Current osteoporosis therapies have major drawbacks (e.g., estrogens increase breast cancer risks), and reduce fractures by only ~20-30%. Most osteoporosis drugs block bone resorption, and there is an urgent need to develop anabolic therapies that can stimulate bone formation. We previously showed that the NO/cGMP/PKG pathway mediates pro-proliferative effects of mechanical stimulation in osteoblasts and anti-apoptotic effects of estrogens in osteocytes [1,2]. These findings lead us to hypothesize that cGMP-elevating agents may have boneprotective effects. Results We tested cinaciguat, a prototype soluble guanylate cyclase stimulator, and nitrosylcobinamide (NO-Cbi), a novel NO-donor with anti-oxidant properties, in a mouse model of estrogen deficiency-induced osteoporosis [3]. Compared with sham-operated mice, ovariectomized mice had lower serum cGMP concentrations, which were largely restored to normal by treatment with cinaciguat, NO-Cbi, or low-dose estrogen replacement. Micro-CT analyses of tibiae showed that all three pharmacological interventions significantly improved trabecular bone architecture in ovariectomized animals, with similar effect sizes. Cinaciguat and NO-Cbi reversed ovariectomy-induced osteocyte apoptosis as efficiently as estradiol, and enhanced bone formation parameters in vivo, consistent with in vitro effects on osteoblast proliferation, differentiation, and survival. As previously reported, ovariectomy dramatically increased osteoclast numbers, and this effect was completely reversed by estradiol. NO-Cbi significantly decreased the number of osteoclasts in ovariectomized mice, whereas cinaciguat had only modest effects, suggesting cGMP-independent effects of NO-Cbi in osteoclasts. Conclusion We conclude that estrogen deficiency represents a state of relative NO and cGMP deficiency, and that NO-dependent or NO-independent guanylate cyclase stimulation may represent a novel, anabolic treatment strategy for post-menopausal osteoporosis. These data confirm an important role of NO/cGMP signaling in bone biology.

Non-genomic thyroid hormone signaling through NO/cGMP/PKGII

Author(s): Kalyanaraman, Hema; Schwappacher, Raphaela; Joshua, Jisha; Zhuang, Shunhui; Scott, Brian; Dillmann, Wolfgang; Frangos, John; Pilz, Renate B