Case of subcutaneous insulin resistance syndrome treated with ultra-rapid insulin lispro.

Subcutaneous insulin resistance syndrome is a rare condition that causes difficulty in glycemic control due to severe resistance to subcutaneous insulin injections. We herein present a case of a 40-year-old woman with type 2 diabetes mellitus who had been diagnosed with subcutaneous insulin resistance syndrome since the age of 29 years, and had been persistently treated with continuous subcutaneous insulin infusion using a mixture of insulin lispro and heparin. The patient was switched from insulin lispro plus heparin to ultra-rapid insulin lispro; given that it contains treprostinil and citrate, it is expected to have similar effects as heparin, and shows similar glucose-lowering effects and insulin absorption. Our results suggest that treatment with ultra-rapid insulin lispro is effective for subcutaneous insulin resistance syndrome.

Euglycemic diabetic ketoacidosis in a patient with type 2 diabetes mellitus 3 days after initiating sodium-glucose cotransporter 2 inhibitor while on an extremely low carbohydrate diet: A case report.

This paper presents a case with type 2 diabetes mellitus on a very-low-carbohydrate diet who developed euglycemic diabetic ketoacidosis (EDKA) 3 days after starting sodium-glucose cotransporter 2 inhibitors (SGLT2i). When initiating SGLT2i, healthcare providers should confirm the implementation of a low-carbohydrate diet and provide intensive guidance to prevent EDKA.

Differential regulation of steroidogenesis by bone morphogenetic proteins in granulosa cells: involvement of extracellularly regulated kinase signaling and oocyte actions in follicle-stimulating hormone-induced estrogen production.

In the present study, we investigated the cellular mechanism by which oocytes and bone morphogenetic proteins (BMPs) govern FSH-induced steroidogenesis using rat primary granulosa cells. BMP-6 and BMP-7 both inhibited FSH- and forskolin (FSK)-induced progesterone synthesis and reduced cAMP synthesis independent of the presence or absence of oocytes. BMP-7 also increased FSH-induced estradiol production, and the response was further augmented in the presence of oocytes. In contrast, BMP-6 had no impact on estradiol synthesis regardless of the presence of oocytes. Because BMP-7 changed neither FSK- nor cAMP-induced estradiol production, the BMP-7 action was mediated through a FSH receptor signaling mechanism that was independent of cAMP-protein kinase A pathway. Treatment with FSH but not cAMP activated ERK1/2 phosphorylation in granulosa cells, which was further accelerated by oocytes. A specific ERK inhibitor, U0126, increased estradiol production and decreased FSH- and FSK-induced progesterone production and cAMP synthesis. This suggests that ERK activation is directly linked to inhibition of estradiol synthesis and amplification of cAMP. Moreover, FSH-induced ERK1/2 phosphorylation was inhibited by BMP-7 but not influenced by BMP-6. In contrast, BMP signaling including Smad1/5/8 phosphorylation and Id-1 transcription was up-regulated by FSH and oocytes in granulosa cells through inhibition of Smad6/7 expression. Collectively, oocytes enhance FSH-induced MAPK activation and BMP signaling in granulosa cells, which leads to differential regulation of steroidogenesis elicited by BMPs in the presence of FSH in developing follicles.

Effects of peroxisome proliferator-activated receptor activation on gonadotropin transcription and cell mitosis induced by bone morphogenetic proteins in mouse gonadotrope LbetaT2 cells.

Involvement of peroxisome proliferator-activated receptor-gamma (PPAR-gamma ) activation and bone morphogenetic protein (BMP) signaling in regulating cell proliferation and hormonal production of pituitary tumors has been reported, although the underlying mechanism remains poorly understood. Here, we investigated regulatory roles of PPARalpha and PPARgamma in gonadotropin transcription and cell mitosis modulated by pituitary activin/BMP systems using a mouse gonadotropinoma cell line Lbeta T2, which expresses activin/BMP receptors, transcription factor Smads, PPARalpha , and PPARgamma . In Lbeta T2 cells, BMP signaling shown by Smad1/5/8 phosphorylation and Id-1 transcription was readily activated by BMPs. A PPARgamma agonist, pioglitazone significantly reduced BMP-induced DNA synthesis by Lbeta T2; whereas the PPARalpha agonist, fenofibric acid, did not. In accordance with the effects on cell mitosis, pioglitazone but not fenofibric acid significantly decreased BMP-induced Id-1-Luc activation. Neither fenofibric acid nor pioglitazone affected activin signaling detected by (CAGA)9-Luc activity. Both PPARalpha and PPARgamma ligands directly suppressed transcriptional activities of FSHbeta , LHbeta , and GnRHR. Activation of PPARalpha and PPARgamma increased mRNA levels of follistatin, but did not affect the expression of follistatin-related gene. Thus, PPAR agonists not only directly suppress gonadotropin transcription and BMP signaling, but also inhibit the biological actions of activins which facilitate gonadotropin transcription through upregulating follistatin expression. In addition, pioglitazone increased BMP ligands mRNA, but decreased activin-beta B mRNA in Lbeta T2 cells. Collectively, PPAR activation differentially regulates gonadotrope cell proliferation and gonadotropin transcription in a ligand-dependent manner.

Regulatory expression of bone morphogenetic protein-6 system in aldosterone production by human adrenocortical cells.

Bone morphogenetic protein-6 (BMP-6) enhances aldosterone production by upregulating angiotensin II (Ang II)-to-MAPK pathway. Here we investigated effects of Ang II and potassium on the BMP system in human adrenocortical H295R cells. BMP-6 transcription was transiently downregulated by treatments with Ang II and potassium. Aldosterone also decreased BMP-6 expression at a high concentration. Chemical inhibitions of transcription and translation abolished the transient reduction of BMP-6, suggesting that destabilization of BMP-6 mRNA was hardly involved while new protein synthesis was possibly mediated in this mechanism. However, BMP-6 protein was stably detected during the exposures of Ang II and potassium. Notably, Ang II, potassium and aldosterone decreased mRNA levels of follistatin that extracellularly neutralizes bioactivities of activins and BMPs although the BMP-6 receptor expression was unaffected. Given the maintenance of bioavailable BMP-6 protein and the receptor expression in adrenocortical cells, endogenous BMP-6 may be a key autocrine modulator for aldosterone production.

Antithyroid drug-associated MPO-ANCA-positive tubulointerstitial nephritis in a type 2 diabetes patient: a case report.

A 54-year-old man diagnosed with type 2 diabetes and hyperthyroidism was prescribed propylthiouracil (PTU) after the patient developed hepatic dysfunction on thiamazole. At 50 mg/day of PTU, he was stable with thyroid-stimulating hormone receptor and thyrotropic antibody titers remaining stable. After four years of taking PTU, he was referred to the Department of Nephrology due to a rapid increase in his serum creatinine (Cr) level. He showed impaired renal function (Cr 2.26 mg/dL; estimated glomerular filtration rate (eGFR), 25 mL/min). In addition, urinary ß2-microglobulin (ß2 MG) was increased to 71,980 µg/L and was positive for myeloperoxidase (MPO)-anti-neutrophil cytoplasmic antibody (ANCA) (33.9 U/mL). Gallium scintigraphy demonstrated a remarkable accumulation in both kidneys. The patient was diagnosed with tubulointerstitial nephritis based on a renal biopsy, the results of which suggested that it might have been induced by PTU. He was treated with prednisolone (PSL) at 30 mg/day. As a result, within two weeks, Cr, eGFR, and urinary ß2 MG levels were progressively improved to 1.72 mg/dL, 34 mL/min, and 22,020 µg/L, respectively. Therefore, we tapered off the PSL with a dose of 5 mg/day after approximately one year. There have been no exacerbated renal function parameters. Although there are many reports on patients developing MPO-ANCA-positive crescentic glomerulonephritis after the administration of PTU, we report on a relatively rare case in which interstitial nephritis occurred after the administration of PTU.

Involvement of bone morphogenetic protein-6 in differential regulation of aldosterone production by angiotensin II and potassium in human adrenocortical cells.

Aldosterone production is modified by several growth factors that reside in the adrenal. We have recently reported the existence of a bone morphogenetic protein (BMP) system in human adrenocortical cells, in which BMP-6 augments aldosterone synthesis. Here, we investigated functional roles of BMP-6, focusing on the differential regulation of aldosterone synthesis induced by angiotensin (Ang) II and potassium (K). In human adrenocortical H295R cells, BMP-6 augmented Ang II-induced CYP11B2 transcription and mRNA and aldosterone production but had no effect on K-induced aldosterone production. Inhibition of endogenous BMP-6 action by neutralizing antibodies impaired aldosterone production induced by Ang II but not that induced by K. Blockage of ligand-receptor binding using extracellular domain (ECD) of BMP type I receptors revealed that ECDs to activin receptor-like kinase (ALK)-2 and ALK-3 significantly reduced the aldosterone production induced by Ang II. None of the type I-receptor ECDs tested had any effect on K-induced aldosterone levels. Overexpression of a dominant negative-activin type II receptor construct selectively decreased Ang II-induced aldosterone production without having any effect on K-induced aldosterone production. BMP type II receptor-dominant negative had no effect on aldosterone induced by either Ang II or K. These results infer that BMP-6 acts through ALK-2, ALK-3, and activin type II receptor receptors in adrenocortical cells. BMP-6 pretreatment extends the induction of ERK1/2 phosphorylation by Ang II and treatment with ECDs to ALK-2 and ALK-3 impaired Ang II-induced ERK phosphorylation. The specific inhibitor of ERK activation, U0126, suppressed the activation of CYP11B2 transcription induced by BMP-6 without affecting Smad phosphorylation and Tlx2-Luc activity. Collectively, the endogenous BMP-6 system plays critical roles in aldosterone production between Ang II and K through ERK signaling pathway.

Regulatory roles of bone morphogenetic proteins and glucocorticoids in catecholamine production by rat pheochromocytoma cells.

We here report a new physiological system that governs catecholamine synthesis involving bone morphogenetic proteins (BMPs) and activin in the rat pheochromocytoma cell line, PC12. BMP type I receptors, including activin receptor-like kinase-2 (ALK-2) (also referred to as ActRIA) and ALK-3 (BMPRIA), both type II receptors, ActRII and BMPRII, as well as the ligands BMP-2, -4, and -7 and inhibin/activin subunits were expressed in PC12 cells. PC12 cells predominantly secrete dopamine, whereas noradrenaline and adrenaline production is negligible. BMP-2, -4, -6, and -7 and activin A each suppressed dopamine and cAMP synthesis in a dose-dependent fashion. The BMP ligands also decreased 3,4-dihydroxyphenylalanine decarboxylase mRNA expression, whereas activin suppressed tyrosine hydroxylase expression. BMPs induced both Smad1/5/8 phosphorylation and Tlx2-Luc activation, whereas activin stimulated 3TP-Luc activity and p38 MAPK phosphorylation. ERK signaling was not affected by BMPs or activin. Dexamethasone enhanced catecholamine synthesis, accompanying increases in tyrosine hydroxylase and 3,4-dihydroxyphenylalanine decarboxylase transcription without cAMP accumulation. In the presence of dexamethasone, BMPs and activin failed to reduce dopamine as well as cAMP production. In addition, dexamethasone modulated mitotic suppression of PC12 induced by BMPs in a ligand-dependent manner. Furthermore, intracellular BMP signaling was markedly suppressed by dexamethasone treatment and the expression of ALK-2, ALK-3, and BMPRII was significantly inhibited by dexamethasone. Collectively, the endogenous BMP/activin system plays a key role in the regulation of catecholamine production. Controlling activity of the BMP system may be critical for glucocorticoid-induced catecholamine synthesis by adrenomedullar cells.

An extra-adrenal abdominal pheochromocytoma causing ectopic ACTH syndrome.

We report a 55-year-old woman with ectopic adrenocorticotropin (ACTH) secretion caused by extra-adrenal pheochromocytoma. The patient presented with a 6-month history of hypertension and diabetes mellitus. Her serum and urinary cortisol levels were extremely high and dexamethasone failed to suppress the cortisol secretion. Her plasma ACTH levels were also elevated (>300 pg/mL) and irresponsive to corticotropin-releasing hormone (CRH) or metyrapone administration. Gel filtration analysis of the patient's plasma detected the existence of large molecular weight ACTH being eluted with a major peak of authentic 1-39 ACTH. Abdominal computed tomographic scan and magnetic resonance imaging revealed a 5-cm paraganglioma located underneath the left kidney, in which (123)I-MIBG tracer specifically accumulated. Bilateral adrenal glands were diffusely enlarged. After surgical removal of the paraganglioma, the patient's clinical symptoms improved and biochemistry normalized including plasma ACTH, urinary free cortisol, and urinary catecholamines. Subsequent histologic evaluation of the transected paranglioma tissue revealed ACTH, synaptin, and chromogranin-A histologically immunostaining. Culture of primary cells collected from the resected paraganglioma demonstrated of in vitro production of ACTH, noradrenaline, and adrenaline. This is the first report of ectopic ACTH syndrome induced by an extra-adrenal abdominal paraganglioma.

Novel action of activin and bone morphogenetic protein in regulating aldosterone production by human adrenocortical cells.

We have uncovered a functional bone morphogenetic protein (BMP) and activin system complete with ligands (BMP-6 and activin betaA/betaB), receptors (activin receptor-like kinase receptors 2, 3, and 4; activin type-II receptor; and BMP type-II receptor), and the binding protein follistatin in the human adrenocortical cell line H295R. Administration of activin and BMP-6 to cultures of H295R cells caused concentration-responsive increases in aldosterone production. The mRNA levels of steroidogenic acute regulatory protein or P450 steroid side-chain cleavage enzyme, the rate-limiting steps of adrenocortical steroidogenesis, were enhanced by activin and BMP-6. Activin and BMP-6 also activated the transcription of steroidogenic acute regulatory protein as well as the late-step steriodogenic enzyme CYP11B2. Activin enhanced ACTH-, forskolin-, or dibutyryl-cAMP- but not angiotensin II (Ang II)-induced aldosterone production, whereas BMP-6 specifically augmented Ang II-induced aldosterone production. Activin and ACTH but not BMP-6 increased cAMP production. Follistatin, which inhibits activin actions by binding, suppressed basal and ACTH-induced aldosterone secretion but failed to affect the Ang II-induced aldosterone level. Furthermore, MAPK signaling appeared to be involved in aldosterone production induced by Ang II and BMP-6 because an inhibitor of MAPK activation, U0126, reduced the level of aldosterone synthesis stimulated by Ang II and BMP-6 but not activin. In addition, Ang II reduced the expression levels of BMP-6 but increased that of activin betaB, whereas ACTH had no effect on these levels. Collectively, the present data suggest that activin acts to regulate adrenal aldosterone synthesis predominantly by modulating the ACTH-cAMP-protein kinase A signaling cascade, whereas BMP-6 works primarily by modulating the Ang II-MAPK cascade in human adrenal cortex in an autocrine/paracrine fashion.

Characterization of the bone morphogenetic protein (BMP) system in human pulmonary arterial smooth muscle cells isolated from a sporadic case of primary pulmonary hypertension: roles of BMP type IB receptor (activin receptor-like kinase-6) in the mitotic action.

The functional involvement of bone morphogenetic protein (BMP) system in primary pulmonary hypertension (PPH) remains unclear. Here we demonstrate a crucial role of the BMP type IB receptor, activin receptor-like kinase (ALK)-6 for pulmonary arterial smooth muscle cell (pphPASMC) mitosis isolated from a sporadic PPH patient bearing no mutations in BMPR2 gene. A striking increase in the levels of ALK-6 mRNA was revealed in pphPASMC compared with control PASMCs, in which ALK-6 transcripts were hardly detectable. BMP-2 and -7 stimulated the mitosis of pphPASMCs, which was opposite to their suppressive effects on the mitosis of the control PASMCs. BMP-4 and -6 and activin inhibited pphPASMC mitosis, whereas these did not affect control PASMCs. The presence of BMP signaling machinery in pphPASMCs was elucidated based on the analysis on Id-1 transcription and Smad-reporter genes. Overexpression of a dominant-negative ALK-6 construct revealed that ALK-6 plays a key role in the mitosis as well as intracellular BMP signaling of pphPASMCs. Gene silencing of ALK-6 using small interfering RNA also reduced DNA synthesis as well as Id-1 transcription in pphPASMCs regardless of BMP-2 stimulation. Although Id-1 response was not stimulated by BMP-2 in control PASMCs, the gene delivery of wild-type ALK-6 caused significant increase in the Id-1 transcripts in response to BMP-2. Additionally, inhibitors of ERK and p38 MAPK pathways suppressed pphPASMC mitosis induced by BMP-2, implying that the mitotic action is in part MAPK dependent. Thus, the BMP system is strongly involved in pphPASMC mitosis through ALK-6, which possibly leads to activation of Smad and MAPK, resulting in the progression of vascular remodeling of pulmonary arteries in PPH.

Role of bradykinin in renoprotective effects by angiotensin II type 1 receptor antagonist in salt-sensitive hypertension.

To elucidate whether bradykinin is involved in the renoprotective effect produced by angiotensin II type 1 receptor antagonist (AT1A) in chronic salt-sensitive hypertension, Dahl salt-sensitive rats receiving a high-salt (8%) diet were treated either with an AT1A (candesartan, 1 mg/kg/day), a bradykinin B2 receptor antagonist (BKB2A; FR172357, 30 mg/kg/day) or a combination of AT1A and BKB2A for 7 weeks. None of the treatments changed the markedly increased systolic blood pressure induced by a high-salt diet. However, chronic treatment with AT1A significantly improved the histological hallmarks of renal damage-i.e., glomerular sclerosis and cell proliferation-despite the presence of severe hypertension. This beneficial action of AT1A was abolished by the concomitant administration of BKB2A. In agreement with these histologically based findings, increases in levels of creatinine clearance induced by AT1A were also reversed back to the basal levels when BKB2A was administered in conjunction with AT1A. Furthermore, urinary excretions of nitrate plus nitrite and prostaglandin E2 increased moderately in response to the administration of AT1A alone, but not in combination with BKB2A. Thus, the blockade of bradykinin signaling abrogates the renoprotective actions of the angiotensin II type 1 (AT1) receptor antagonism. Collectively, these data show that when AT1 action is chronically blocked, endogenous bradykinin plays a pivotal role in preventing the progression of glomerular injury in salt-sensitive hypertension.

Involvement of bone morphogenetic protein activity in somatostatin actions on ovarian steroidogenesis.

Somatostatin is expressed in the hypothalamus, pancreas and gastrointestinal tracts and it inhibits the secretion of various hormones in vivo. In the rodent ovary, somatostatin receptor (SSTR) subtypes 2 and 5 are expressed in granulosa cells and oocytes. Somatostatin analogs have been clinically used for treatment of endocrine tumors. For this purpose, relatively high-dose or long-term treatments of somatostatin analogs are necessary; however, the direct and continuous impact of somatostatin analogs on gonadal functions has yet to be elucidated. In the present study, we investigated the effects of somatostatin analogs (octreotide and pasireotide) on ovarian steroidogenesis by rat primary granulosa cell culture. The expression levels of SSTR2 and SSTR5 in granulosa cells were upregulated by FSH treatment. Treatment with somatostatin analogs decreased FSH-induced estradiol production with reduction in aromatase mRNA expression, while the treatment also suppressed FSH-induced progesterone production with reduction of mRNAs levels of StAR, P450scc and 3ßHSD2 in granulosa cells. This trend was also observed in a granulosa/oocyte co-culture condition. The effect of pasireotide was more potent than that of octreotide. FSH-induced synthesis of steroids and cAMP was also suppressed by somatostatin analog treatment. Notably, pretreatment with a BMP-binding protein, noggin reversed the suppressive effects of somatostatin analogs on progesterone and cAMP production, suggesting that the endogenous BMP system is functionally involved in the SSTR effects in granulosa cells. Treatment with BMP-2, -4, -6 and -7 decreased the mRNA expression of inhibitory Smads6 and 7, leading to enhancement of BMP actions detected by Id-1 transcription in granulosa cells. Collectively, the results revealed that SSTR activation modulates ovarian steroidogenesis by upregulating endogenous BMP activity in growing follicles.

Rapid progression of Graves' ophthalmopathy despite the administration of thiamazole.

A 42-year-old female with body weight loss, finger tremors and ocular discomfort was diagnosed with Graves' disease complicated with ophthalmopathy. Thiamazole therapy rapidly improved her hyperthyroidism. However, she was admitted to our hospital because her eye symptoms acutely deteriorated over a period of two weeks. She had ocular immotility, exposure keratitis, conjunctival edema, severe proptosis and visual impairment with a high titer of serum thyroid-stimulating antibody (TSAb). Methylprednisolone pulse therapy at a dose of 500 mg/day improved her eye symptoms. Although the mechanism of the progression of Graves' ophthalmopathy has not yet been elucidated, special attention should be paid to the occurrence of ophthalmopathy even after the initiation of thiamazole therapy.

Interaction between gonadotropin-releasing hormone and bone morphogenetic protein-6 and -7 signaling in LßT2 gonadotrope cells.

It is known that bone morphogenetic proteins (BMPs) regulate gonadotropin transcription and production by pituitary gonadotrope cells. However, the role of BMPs in gonadotropin-releasing hormone (GnRH)-induced FSH production remains uncertain. Here, we describe a functional link between BMP-6 and BMP-7 signals and FSH transcriptional activity induced by GnRH using mouse gonadotrope LßT2 cells. In LßT2 cells, BMP-6 and BMP-7 increased mouse FSHß-promoter activity in a concentration-dependent manner. The induction by BMP-6 and BMP-7 was inhibited by treatment with extracellular domains of ActRII but not BMPRII. These findings suggest that the type II receptor ActRII participates in BMP-induced FSHß transcription regulation. Notably, BMP-6, but not BMP-7, enhanced GnRH-induced FSHß-promoter activity in LßT2 cells. Since GnRH stimulated MAPK phosphorylation in LßT2 cells, a functional link between MAPK and FSHß transcription was examined. Inhibition of the ERK pathway, but not that of p38 or SAPK/JNK signaling, suppressed GnRH-induced FSHß transcription, suggesting that ERK is functionally involved in GnRH-induced FSHß transcription. Co-treatment with BMP-7, but not with BMP-6, suppressed GnRH-induced MAPK phosphorylation in LßT2 cells. Thus, the difference between BMP-6 and BMP-7 in enhancing GnRH-induced FSHß transcription may be due to the differential effects of BMP ligands on GnRH-induced ERK signaling. On the other hand, GnRH reduced Smad1/5/8 phosphorylation but increased Smad6/7 expression. These findings imply the presence of a functional link between GnRH action, MAPK signaling and the BMP system in pituitary gonadotropes for fine-tuning of FSH gene expression.

Regulatory role of kit ligand-c-kit interaction and oocyte factors in steroidogenesis by rat granulosa cells.

Although kit ligand (KL)-c-kit interaction is known to be critical for oogenesis and folliculogenesis, its role in ovarian steroidogenesis has yet to be elucidated. We studied the impact of KL-c-kit interaction in regulation of steroidogenesis using rat oocyte/granulosa cell co-culture. In the presence of oocytes, soluble KL suppressed FSH-induced estradiol production and aromatase mRNA expression without affecting FSH-induced progesterone production. The KL effect on steroidogenesis was interrupted by an anti-c-kit neutralizing antibody, suggesting that KL-c-kit interaction is involved in suppression of estrogen by granulosa cells through oocyte c-kit action. The cAMP-PKA pathway activity was not directly involved in the estrogen regulation by KL-c-kit action. It was of note that KL treatment increased the expression levels of oocyte-derived FGF-8, GDF-9 and BMP-6, while it reduced the expression levels of oocyte-derived BMP-15 in the oocyte-granulosa cell co-culture. Given the findings that FGF-8, but not GDF-9, BMP-6 or -15, suppressed FSH-induced estrogen production by granulosa cells, oocyte-derived FGF-8 is linked to suppression of FSH-induced estrogen production through the KL-c-kit interaction. Furthermore, the suppression of FSH-induced estrogen production by KL in the co-culture was reversed by a FGF receptor kinase inhibitor and the effect of the inhibitor was enhanced in combination with extracellular-domain protein of BMPRII, which interferes with BMP-15 and GDF-9 activities. Thus, the actions of endogenous oocyte factors including FGF-8 and BMP-15/GDF-9 were involved in the KL activity that inhibited FSH-induced estradiol production. Collectively, the results indicate that KL-c-kit interaction plays a role in estrogenic regulation through oocyte-granulosa cell communication.

Peroxisome proliferator-activated receptor activity is involved in the osteoblastic differentiation regulated by bone morphogenetic proteins and tumor necrosis factor-α.

Recent studies have suggested possible adverse effects of thiazolidinediones on bone metabolism. However, the detailed mechanism by which the activity of PPAR affects bone formation has not been elucidated. Impaired osteoblastic function due to cytokines is critical for the progression of inflammatory bone diseases. In the present study, we investigated the cellular mechanism by which PPAR actions interact with osteoblast differentiation regulated by BMP and TNF-α using mouse myoblastic C2C12 cells. BMP-2 and -4 potently induced the expression of various bone differentiation markers including Runx2, osteocalcin, type-1 collagen and alkaline phosphatase (ALP) in C2C12 cells. When administered in combination with a PPARα agonist (fenofibric acid) but not with a PPARγ agonist (pioglitazone), BMP-4 enhanced osteoblast differentiation through the activity of PPARα. The osteoblastic changes induced by BMP-4 were readily suppressed by treatment with TNF-α. Interestingly, the activities of PPARα and PPARγ agonists reversed the suppression by TNF-α of osteoblast differentiation induced by BMP-4. Furthermore, TNF-α-induced phosphorylation of MAPKs, NFκB, IκB and Stat pathways was inhibited in the presence of PPARα and PPARγ agonists with reducing TNF-α receptor expression. In view of the finding that inhibition of SAPK/JNK, Stat and NFκB pathways reversed the TNF-α suppression of osteoblast differentiation, we conclude that these cascades are functionally involved in the actions of PPARs that antagonize TNF-α-induced suppression of osteoblast differentiation. It was further discovered that the PPARα agonist enhanced BMP-4-induced Smad1/5/8 signaling through downregulation of inhibitory Smad6/7 expression, whereas the PPARγ agonist impaired this activity by suppressing BMPRII expression. On the other hand, BMPs increased the expression levels of PPARα and PPARγ in the process of osteoblast differentiation. Thus, PPARα actions promote BMP-induced osteoblast differentiation, while both activities of PPARα and PPARγ suppress TNF-α actions. Collectively, our present data establishes that PPAR activities are functionally involved in modulating the interaction between the BMP system and TNF-α receptor signaling that is crucial for bone metabolism.

Enhanced expression of bone morphogenetic protein system in aldosterone-treated mouse kidneys.

Recent studies have shown that bone morphogenetic proteins (BMPs), particularly BMP-7, have an inhibitory role in the development of various renal diseases. We previously reported antagonistic effects of BMPs on renal mesangial cell proliferation induced by aldosterone (Aldo) in vitro. In the present study, we investigated in vivo roles of BMPs in Aldo-induced renal glomerular injury. BALB/c mice aged 6 weeks were treated with Aldo injection (5 µg per day, intraperitoneally) and/or oral administration of high-salt (2%) water for 9 weeks. Systemic blood pressure, body weight, kidney weight and daily proteinuria were not significantly changed by Aldo and/or high-salt treatment. However, renal histological examination revealed increases in glomerular cellularity and glomerular diameter in the groups treated with Aldo injection and high-salt administration. Immunohistochemistry demonstrated expression of BMP-4 and -7 in the glomerular mesangial region. Aldo causes renal glomerular damage by stimulating mesangial cell proliferation and increasing extracellular matrix via the mineralocorticoid receptor (MR). MR messenger RNA (mRNA) expression in the renal cortex was transiently increased by 3-week treatment with Aldo and high-salt intake, but was decreased by 9-week treatment. Furthermore, the expression levels of BMP-4 and -7 mRNA were enhanced in the renal cortex treated with Aldo and high-salt administration. These findings suggest that the renal BMP system is activated by Aldo under the condition of high-salt exposure, which may have a key role in antagonizing glomerular damage in vivo.

An in vivo role of bone morphogenetic protein-6 in aldosterone production by rat adrenal gland.

Aldosterone is synthesized in the zona glomerulosa of the adrenal cortex. We previously reported the presence of a functional BMP system including BMP-6 in human adrenocortical cells. BMP-6 contributes to Ang II-induced aldosterone production by activating Smad signaling, in which endogenous BMP-6 action is negatively controlled by Ang II in vitro. In the present study, we examined the in vivo role of BMP-6 in regulation of aldosterone by neutralizing endogenous BMP-6 in rats treated with immunization against BMP-6. Three-week-old male rats were actively immunized with rat mature BMP-6 antigen conjugated with keyhole limpet hemocyanin (KLH). The immunization treatment had no effect on bilateral adrenal weight or its ratio to body weight. Urinary aldosterone excretion was time-dependently increased during the 8-week observation period in the control group. Of note, the level of urinary aldosterone excretion in BMP-6-KLH-immunized rats was significantly reduced compared to that in the control group, suggesting that endogenous BMP-6 contributes to the induction of aldosterone production in vivo. Moreover, the level of urinary aldosterone/creatinine after 8-week treatment was significantly lowered by treatment with BMP-6-KLH. In contrast, with chronic Ang II treatment, urinary aldosterone and creatinine-corrected values at 8 weeks were not significantly different between the two groups, suggesting that the effects of BMP-6-KLH were impaired under the condition of chronic treatment with Ang II. The mRNA levels of Cyp11b2, but not those of Star, P450scc and 3ßhsd2, were significantly decreased in adrenal tissues isolated from BMP-6-KLH-immunized rats after 8-week treatment. Furthermore, the ratio of plasma aldosterone level to corticosterone was significantly decreased by immunization with BMP-6-KLH. Collectively, the results indicate that endogenous BMP-6 is functionally linked to aldosterone synthesis by the zona glomerulosa in the adrenal cortex in vivo.

Mutual regulation of growth hormone and bone morphogenetic protein system in steroidogenesis by rat granulosa cells.

GH induces preantral follicle growth and differentiation with oocyte maturation. However, the effects of GH on ovarian steroidogenesis and the mechanisms underlying its effects have yet to be elucidated. In this study, we investigated the actions of GH on steroidogenesis by rat granulosa cells isolated from early antral follicles by focusing on the ovarian bone morphogenetic protein (BMP) system. We found that GH suppressed FSH-induced estradiol production with reduction in aromatase expression and, in contrast, GH increased FSH-induced progesterone level with induction of steroidogenic acute regulatory protein, side chain cleavage cytochrome P450, and 3ß-hydroxysteroid dehydrogenase. The effects of GH on steroidogenesis by granulosa cells were enhanced in the presence of the BMP antagonist noggin. Coculture of GH with oocytes did not alter GH regulation of steroidogenesis. Steroid production induced by cAMP donors was not affected by GH treatment and the GH effects on FSH-induced steroid production were not accompanied by changes in cAMP synthesis, suggesting that GH actions were not directly mediated by the cAMP-protein kinase A pathway. GH exerted synergistic effects on MAPK activation elicited by FSH, which regulated FSH-induced steroidogenesis. In addition, GH-induced signal transducer and activator of transcription phosphorylation was involved in the induction of IGF-I expression. GH increased IGF-I, IGF-I receptor, and FSH receptor expression in granulosa cells, and inhibition of IGF-I signaling restored GH stimulation of FSH-induced progesterone production, suggesting that endogenous IGF-I is functionally involved in GH effects on progesterone induction. BMP inhibited IGF-I effects that increased FSH-induced estradiol production with suppression of expression of the GH/IGF-I system, whereas GH/IGF-I actions impaired BMP-Sma and Mad related protein 1/5/8 signaling through down-regulation of the expression of BMP receptors. Thus, GH acts to modulate estrogen and progesterone production differentially through endogenous IGF-I activity in granulosa cells, in which GH-IGF-I interaction leads to antagonization of BMP actions including suppression of FSH-induced progesterone production. Mutual balance between GH/IGF-I and BMP signal intensities may be a key for regulating gonadotropin-induced steroidogenesis in growing follicles.