Noriko Makita

Mechanical stress activates angiotensin II type 1 receptor without the involvement of angiotensin II

The angiotensin II type 1 (AT1) receptor has a crucial role in load-induced cardiac hypertrophy. Here we show that the AT1 receptor can be activated by mechanical stress through an angiotensin-II-independent mechanism. Without the involvement of angiotensin II, mechanical stress not only activates extracellular-signal-regulated kinases and increases phosphoinositide production in vitro, but also induces cardiac hypertrophy in vivo. Mechanical stretch induces association of the AT1 receptor with Janus kinase 2, and translocation of G proteins into the cytosol. All of these events are inhibited by the AT1 receptor blocker candesartan. Thus, mechanical stress activates AT1 receptor independently of angiotensin II, and this activation can be inhibited by an inverse agonist of the AT1 receptor.

An acquired hypocalciuric hypercalcemia autoantibody induces allosteric transition among active human Ca-sensing receptor conformations.

The seven-spanning calcium-sensing receptor (CaSR) activates multiple G proteins including Gq and Gi, and thereby activates a variety of second messengers and inhibits parathyroid hormone (PTH) secretion. However, the exact signaling mechanisms underlying the functional activity of CaSR are not yet fully understood. The heterozygous inactivation of CaSR or its inhibition by antibody blocking results in either familial hypocalciuric hypercalcemia or acquired hypocalciuric hypercalcemia (AHH), respectively. Here, we report the identification of a unique CaSR autoantibody in an AHH patient. Paradoxically, we find that this autoantibody potentiates the Ca2+/Gq-dependent accumulation of inositol phosphates by slightly shifting the dose dependence curve of the Ca2+ mediated activation of phosphatidylinositol turnover to the left, whereas it inhibits the Ca2+/Gi-dependent phosphorylation of ERK1/2 in HEK293 cells stably expressing human CaSR. Treatment of these same cells with a calcimimetic, NPS-R-568, augments the CaSR response to Ca2+, increasing phosphatidylinositol turnover and ERK1/2 phosphorylation, and overcoming the autoantibody effects. Our observations thus indicate that a calcium-stimulated CaSR primed by a specific autoantibody adopts a unique conformation that activates Gq but not Gi. Our findings also suggest that CaSR signaling may act via both Gq and Gi to inhibit PTH secretion. This is the first report of a disease-related autoantibody that functions as an allosteric modulator and maintains G protein-coupled receptors (GPCRs) in a unique active conformation with its agonist. We thus speculate that physiological modulators may exist that enable an agonist to specifically activate only one signaling pathway via a GPCR that activates multiple signaling pathways.

Distinct Roles of Smad2-, Smad3-, and ERK-dependent Pathways in Transforming Growth Factor-β1 Regulation of Pancreatic Stellate Cellular Functions

Pancreatic stellate cells (PSCs) play a major role in promoting pancreatic fibrosis. Transforming growth factor-β1 (TGF-β1) regulates PSC activation and proliferation in an autocrine manner. The intracellular signaling pathways of the regulation were examined in this study. Immunoprecipitation and immunocytochemistry revealed that Smad2, Smad3, and Smad4 were functionally expressed in PSCs. Adenovirus-mediated expression of Smad2, Smad3, or dominant-negative Smad2/3 did not alter TGF-β1 mRNA expression level or the amount of autocrine TGF-β1 peptide. However, expression of dominant-negative Smad2/3 inhibited PSC activation and enhanced their proliferation. Co-expression of Smad2 with dominant-negative Smad2/3 restored PSC activation inhibited by dominant-negative Smad2/3 expression without changing their proliferation. By contrast, co-expression of Smad3 with dominant-negative Smad2/3 attenuated PSC proliferation enhanced by dominant-negative Smad2/3 expression without altering their activation. Exogenous TGF-β1 increased TGFβ1 mRNA expression in PSCs. However, PD98059, a specific inhibitor of mitogen-activated protein kinase kinase (MEK1), inhibited ERK activation by TGF-β1, and consequently attenuated TGF-β1 enhancement of its own mRNA expression in PSCs. We propose that TGF-β1 differentially regulates PSC activation, proliferation, and TGF-β1 mRNA expression through Smad2-, Smad3-, and ERK-dependent pathways, respectively.

Sunitinib Induces Hypothyroidism with a Markedly Reduced Vascularity

BACKGROUND Sunitinib is a small molecule that inhibits receptor tyrosine kinases, including the vascular endothelial growth factor receptors, and exhibits antiangiogenic and antitumor activity. This molecule has also been reported to cause hypothyroidism at a high frequency, but the mechanism of this is unknown. SUMMARY A 60-year-old woman was administered sunitinib for the treatment of metastatic renal cell carcinoma. One week later, she displayed overt hypothyroidism with an atrophic thyroid and a marked reduction in vascularity as determined by ultrasonography, despite high levels of thyrotropin. In contrast, during the off-periods in the sunitinib treatment cycles, the volume of her thyroid recovered with an increase in vascularity despite a low level of thyrotropin. These results suggest that thyroid function and volume may depend on the vascularity, which is negatively regulated by sunitinib. CONCLUSION Our case study provides compelling evidence that sunitinib induces hypothyroidism by reducing blood flow via capillary regression and constriction.

Tyrosine Kinase Inhibitor–Induced Thyroid Disorders: A Review and Hypothesis

BACKGROUND Thyroid dysfunction is a well-known adverse effect of sunitinib, a drug that targets multiple receptor tyrosine kinases, including vascular endothelial growth factor receptor (VEGFR). As several kinds of tyrosine kinase inhibitors (TKIs) are now available, this has been postulated to be a side effect of the TKIs that target the VEGFR (VEGF-TKIs). However, sunitinib, one of the first-generation TKIs, likely causes thyroid dysfunction more frequently than other TKI classes, leading not only to hypothyroidism, but also to thyrotoxicosis. SUMMARY Based on the reports published to date, including our own studies, we have hypothesized that sunitinib may exert these effects, because it targets a broad spectrum of tyrosine kinases. This not only includes VEGFR2, but also VEGFR1 and the platelet-derived growth factor receptor (PDGFR). This, in turn, may suggest that not only VEGFR2 but also the PDGFR and/or the VEGFR1 play an important role during angiogenesis in the thyroid. CONCLUSIONS Our current hypothesis may explain the mechanisms that underlie TKI-induced thyroid disorders. By learning how various kinds of TKIs affect thyroid function, we may elucidate how the angiogenesis in thyroid is regulated both physiologically and pathologically.

Lack of evidence for AT1R/B2R heterodimerization in COS-7, HEK293, and NIH3T3 cells: how common is the AT1R/B2R heterodimer?

It has been suggested previously ( AbdAlla, S., Lother, H., and Quitterer, U. (2000) Nature 407, 94-98 ) that the angiotensin II type 1 receptor (AT1R) and the bradykinin B2 receptor (B2R) form constitutive heterodimers. Furthermore they demonstrate that AT1R signaling significantly increases in the presence of the B2R. These findings suggest that heterodimerization and potentiation of AT1R signaling is a universal phenomenon that occurs as a natural consequence of simultaneous expression of the two receptors. Hence this potential interaction is of great pharmacological and biological interest that adds an additional layer of complexity to the understanding of the cross-talk between the renin-angiotensin and kallikrein-kinin systems. Given the remarkable significance of this finding, scientists from four independent research groups have set out to reproduce and further examine the potential AT1R/B2R interaction. We have investigated functional potentiation by the B2R of AT1R signaling in three different cell lines using multiple assays including phosphoinositide hydrolysis, ERK activation, β-arrestin recruitment, and receptor selection and amplification technology, and we have examined dimerization using bioluminescence resonance energy transfer and regulated secretion/aggregation technology. However, although both the AT1Rs and B2Rs were functional in our systems and the systems were fine tuned to detect small changes in receptor function, we failed to detect any functional modulation by or physical interaction between the two receptor proteins. In contrast to the previous observations, our data collectively suggest that AT1R/B2R heterodimerization does not occur as a natural consequence of their simultaneous expression in the same cell nor does the B2R influence the AT1R signaling.

V2 Vasopressin Receptor (V2R) Mutations in Partial Nephrogenic Diabetes Insipidus Highlight Protean Agonism of V2R Antagonists

Background: Inactivating mutations of the V2 receptor (V2R) cause nephrogenic diabetes insipidus (NDI). Results: Two V2R mutants discovered in partial NDI show partial defects, and V2R antagonists rescued them. Conclusion: V2R antagonists operate as pharmacochaperones for defective mutants, whereas they operate as inverse agonists for normal receptors. Significance: V2R antagonists can act as protean agonists, potentially underlying their dual effects. Inactivating mutations of the V2 vasopressin receptor (V2R) cause cross-linked congenital nephrogenic diabetes insipidus (NDI), resulting in renal resistance to the antidiuretic hormone AVP. In two families showing partial NDI, characterized by an apparently normal response to diagnostic tests and an increase in the basal ADH levels suggesting AVP resistance, we have identified two V2R mutations, Ser-333del and Y128S. Both mutant V2Rs, when expressed in COS-7 cells, show partial defects in vasopressin-stimulated cAMP accumulation and intracellular localization. The inhibition of internalization does not rescue their localization. In contrast, the non-peptide V2R antagonists OPC41061 and OPC31260 partially rescue the membrane localization and basal function of these V2R mutants, whereas they inhibit the basal activity of the wild-type V2R. These results indicate that a partial loss of function of Ser-333del and Y128S mutant V2Rs results from defective membrane trafficking. These findings further indicate that V2R antagonists can act as protean agonists, serving as pharmacological chaperones for inactivating V2R mutants and also as inverse agonists of wild-type receptors. We speculate that this protean agonism could underlie the possible dual beneficial effects of the V2R antagonist: improvement of hyponatremia with heart failure or polycystic kidney disease and potential rescue of NDI.

Human Gsα mutant causes pseudohypoparathyroidism type Ia/neonatal diarrhea, a potential cell-specific role of the palmitoylation cycle

Pseudohypoparathyroidism type Ia (PHP-Ia) results from the loss of one allele of Gsα, causing resistance to parathyroid hormone and other hormones that transduce signals via Gs. Most Gsαmutations cause the complete loss of protein expression, but some cause loss of function only, and these have provided valuable insights into the normal function of G proteins. Here we have analyzed a mutant Gsα (αs-AVDT) harboring AVDT amino acid repeats within its GDP/GTP binding site, which was identified in unique patients with PHP-Ia accompanied by neonatal diarrhea. Biochemical and intact cell analyses showed that αs-AVDT is unstable but constitutively active as a result of rapid GDP release and reduced GTP hydrolysis. This instability underlies the PHP-Ia phenotype. αs-AVDT is predominantly localized in the cytosol, but in rat and mouse small intestine epithelial cells (IEC-6 and DIF-12 cells) αs-AVDT was found to be localized predominantly in the membrane where adenylyl cyclase is present and constitutive increases in cAMP accumulation occur in parallel. The likely cause of this membrane localization is the inhibition of an activation-dependent decrease in αs palmitoylation. Upon the overexpression of acyl-protein thioesterase 1, however, αs-AVDT translocates from the membrane to the cytosol, and the constitutive accumulation of cAMP becomes attenuated. These results suggest that PHP-Ia results from the instability of αs-AVDT and that the accompanying neonatal diarrhea may result from its enhanced constitutive activity in the intestine. Hence, palmitoylation may control the activity and localization of Gsα in a cell-specific manner.

Regulation of RhoA Signaling by the cAMP-dependent Phosphorylation of RhoGDIα

Background: cAMP-induced phosphorylation of RhoA has been considered to inhibit RhoA signaling, causing cell rounding. Results: Knockdown of RhoGDIα blocks cAMP-induced cell rounding, and RhoGDIα-WT expression but not RhoGDIα-S174A expression recovers. Conclusion: Phosphorylation of RhoGDIα likely inhibits RhoA by stabilizing a active RhoA-RhoGDIα complex. Significance: This may underlie Gs/cAMP-induced cross-talk with Gq/G13/RhoA signaling. RhoA plays a pivotal role in regulating cell shape and movement. Protein kinase A (PKA) inhibits RhoA signaling and thereby induces a characteristic morphological change, cell rounding. This has been considered to result from cAMP-induced phosphorylation of RhoA at Ser-188, which induces a stable RhoA-GTP-RhoGDIα complex and sequesters RhoA to the cytosol. However, few groups have shown RhoA phosphorylation in intact cells. Here we show that phosphorylation of RhoGDIα but not RhoA plays an essential role in the PKA-induced inhibition of RhoA signaling and in the morphological changes using cardiac fibroblasts. The knockdown of RhoGDIα by siRNA blocks cAMP-induced cell rounding, which is recovered by RhoGDIα-WT expression but not when a RhoGDIα-S174A mutant is expressed. PKA phosphorylates RhoGDIα at Ser-174 and the phosphorylation of RhoGDIα is likely to induce the formation of a active RhoA-RhoGDIα complex. Our present results thus reveal a principal molecular mechanism underlying Gs/cAMP-induced cross-talk with Gq/G13/RhoA signaling.

High serum ALP level is associated with increased risk of denosumab-related hypocalcemia in patients with bone metastases from solid tumors

Metastatic bone disease is one of the most common complications of advanced cancers. Pathological fractures, spinal cord compression, and radiotherapy or surgery to the bone are collectively called skeletal-related events (SREs), which cause severe pain, increase hospitalization rates, and impair the quality of life (QOL) of patients with bone metastases. The receptor activator of nuclear factor-kB ligand (RANKL)/RANK pathway is critical in the progression of bone metastases. Previous studies have demonstrated that an anti-RANKL antibody (denosumab) was superior to zoledronic acid in prolonging time to first SRE in patients with bone metastases from prostate and breast cancers. However, severe hypocalcemic events occur more frequently after treatment with denosumab compared with zoledronic acid. In this study, 368 administrations of denosumab in 219 patients with metastatic bone disease from solid tumors were analyzed to clarify the risk factors for developing hypocalcemia. The results showed that grade 2/3 hypocalcemia was observed in 10.4% of the total number of denosumab administrations. Patients with higher baseline serum ALP, higher performance status (PS), or gastric cancer were at higher risk for developing hypocalcemia. The cut-off value for ALP to predict denosumab-related hypocalcemia was 587 U/L with a sensitivity of 0.77 and a specificity of 0.81. Close monitoring of serum calcium, especially after the first treatment with denosumab, is strongly recommended in these patients.