C/EBPβ dictates postmenopausal FSHβ transcription and blockade of AEP/C/EBPβ pathway alleviates osteoporosis

Abstract

Follicle-stimulating hormone (FSH), a gonadotropin that rises in post-menopausal females, activates its receptor FSHR to trigger bone loss via increasing bone resorption by osteoclasts. FSH stimulates CCAAT/enhancer binding protein beta (C/EBPβ) /asparagine endopeptidase (AEP) pathway, facilitating neural degeneration in the brain of mouse models with Alzheimer’s disease (AD). However, whether C/EBPβ/AEP pathway feeds back and modulates FSHβ bone resorption action remains elusive. Here we show that C/EBPβ acts as a transcription factor for fshb gene and directly binds its promoter, mediating its mRNA transcription in the pituitary gland. Knocking down C/EBPβ in primary pituitary cells significantly blunts GnRH (gonadotropin-releasing hormone)-induced FSHβ expression. Knockout of C/EBPβ also robustly diminishes FSHβ levels in mice. Inactivation of AEP, either by knockout of AEP or its small molecular inhibitor, antagonizes C/EBPβ and suppresses FSHβ levels, attenuating ovariectomy (OVX)-elicited osteoporosis. Markedly, a specific AEP inhibitor (#11a) displays comparable therapeutic effect as an FDA-approved drug teriparatide in OVX-induced osteoporosis. Hence, these findings support that C/EBPβ dictates FSHβ transcription and blocking AEP by its inhibitor represses C/EBPβ-mediated FSHβ levels, exerting prominent therapeutic efficacy toward osteoporosis.

References

[1] Das N, Kumar TR. Molecular regulation of follicle-stimulating hormone synthesis, secretion and action. J. Mol. Endocrinol.. 2018, 60: R131-R155.

[2] Ortolano GAet al. . Follicle-stimulating hormone beta subunit messenger ribonucleic acid concentrations during the rat estrous cycle. Endocrinology. 1988, 123: 2946-2948.

[3] Halvorson LM, Weiss J, Bauer-Dantoin AC, Jameson JL. Dynamic regulation of pituitary follistatin messenger ribonucleic acids during the rat estrous cycle. Endocrinology. 1994, 134: 1247-1253.

[4] Nakamura Tet al. . Activin-binding protein from rat ovary is follistatin. Science. 1990, 247: 836-838.

[5] Bilezikjian LMet al. . Autocrine/paracrine regulation of pituitary function by activin, inhibin and follistatin. Mol. Cell Endocrinol.. 2004, 225: 29-36.

[6] Mason AJet al. . A deletion truncating the gonadotropin-releasing hormone gene is responsible for hypogonadism in the hpg mouse. Science. 1986, 234: 1366-1371.

[7] Taneja Cet al. . FSH-metabolic circuitry and menopause. J. Mol. Endocrinol.. 2019, 63: R73-R80.

[8] Randolph JFJr.et al. . Reproductive hormones in the early menopausal transition: relationship to ethnicity, body size, and menopausal status. J. Clin. Endocrinol. Metab.. 2003, 88: 1516-1522.

[9] Sun Let al. . FSH directly regulates bone mass. Cell. 2006, 125: 247-260.

[10] Liu Pet al. . Blocking FSH induces thermogenic adipose tissue and reduces body fat. Nature. 2017, 546: 107-112.

[11] Zhu LLet al. . Blocking antibody to the beta-subunit of FSH prevents bone loss by inhibiting bone resorption and stimulating bone synthesis. Proc. Natl. Acad. Sci. USA. 2012, 109: 14574-14579.

[12] Silverman E, Eimerl S, Orly J. CCAAT enhancer-binding protein beta and GATA-4 binding regions within the promoter of the steroidogenic acute regulatory protein (StAR) gene are required for transcription in rat ovarian cells. J. Biol. Chem.. 1999, 274: 17987-17996.

[13] LaVoie HA, Singh D, Hui YY. Concerted regulation of the porcine steroidogenic acute regulatory protein gene promoter activity by follicle-stimulating hormone and insulin-like growth factor I in granulosa cells involves GATA-4 and CCAAT/enhancer binding protein beta. Endocrinology. 2004, 145: 3122-3134.

[14] Sirois J, Richards JS. Transcriptional regulation of the rat prostaglandin endoperoxide synthase 2 gene in granulosa cells. Evidence for the role of a cis-acting C/EBP beta promoter element. J. Biol. Chem.. 1993, 268: 21931-21938.

[15] Gronning LMet al. . Isoform-specific regulation of the CCAAT/enhancer-binding protein family of transcription factors by 3’,5’-cyclic adenosine monophosphate in Sertoli cells. Endocrinology. 1999, 140: 835-843.

[16] Niehrs C, Calkhoven CF. Emerging role of C/EBPβ and epigenetic DNA methylation in ageing. Trends Genet.. 2020, 36: 71-80.

[17] Kim Ket al. . MafB negatively regulates RANKL-mediated osteoclast differentiation. Blood. 2007, 109: 3253-3259.

[18] Nerlov C. The C/EBP family of transcription factors: a paradigm for interaction between gene expression and proliferation control. Trends Cell Biol.. 2007, 17: 318-324.

[19] Tominaga Het al. . CCAAT/enhancer-binding protein beta promotes osteoblast differentiation by enhancing Runx2 activity with ATF4. Mol. Biol. Cell. 2008, 19: 5373-5386.

[20] Hata Ket al. . A CCAAT/enhancer binding protein beta isoform, liver-enriched inhibitory protein, regulates commitment of osteoblasts and adipocytes. Mol. Cell Biol.. 2005, 25: 1971-1979.

[21] Li DN, Matthews SP, Antoniou AN, Mazzeo D, Watts C. Multistep autoactivation of asparaginyl endopeptidase in vitro and in vivo. J. Biol. Chem.. 2003, 278: 38980-38990.

[22] Liu Zet al. . Neuroprotective actions of PIKE-L by inhibition of SET proteolytic degradation by asparagine endopeptidase. Mol. Cell. 2008, 29: 665-678.

[23] Zhang Zet al. . Cleavage of tau by asparagine endopeptidase mediates the neurofibrillary pathology in Alzheimer’s disease. Nat. Med.. 2014, 20: 1254-1262.

[24] Zhang Zet al. . Delta-secretase cleaves amyloid precursor protein and regulates the pathogenesis in Alzheimer’s disease. Nat. Commun.. 2015, 6. 8762

[25] Wang ZHet al. . C/EBPbeta regulates delta-secretase expression and mediates pathogenesis in mouse models of Alzheimer’s disease. Nat. Commun.. 2018, 9. 1784

[26] Wang ZHet al. . Neuronal ApoE4 stimulates C/EBPbeta activation, promoting Alzheimer’s disease pathology in a mouse model. Prog. Neurobiol.. 2022, 209. 102212

[27] Xiong Jet al. . FSH blockade improves cognition in mice with Alzheimer’s disease. Nature. 2022, 603: 470-476.

[28] Xiong J, Zhang Z, Ye K. C/EBPbeta/AEP signaling drives Alzheimer’s disease pathogenesis. Neurosci. Bull.. 2023, 39: 1173-1185.

[29] Croucher PI, Garrahan NJ, Compston JE. Assessment of resorption cavity characteristics in trabecular bone: changes in primary and secondary osteoporosis. Bone. 1993, 14: 449-454.

[30] Compston JE, McClung MR, Leslie WD. Osteoporosis. Lancet. 2019, 393: 364-376.

[31] Choi SJet al. . Identification of human asparaginyl endopeptidase (legumain) as an inhibitor of osteoclast formation and bone resorption. J. Biol. Chem.. 1999, 274: 27747-27753.

[32] Xiong Jet al. . A TrkB agonist prodrug prevents bone loss via inhibiting asparagine endopeptidase and increasing osteoprotegerin. Nat. Commun.. 2022, 13. 4820

[33] Wang, Z. H. et al. BDNF inhibits neurodegenerative disease-associated asparaginyl endopeptidase activity via phosphorylation by AKT. JCI Insight3, e99007 (2018).

[34] Bliss SPet al. . ERK signaling in the pituitary is required for female but not male fertility. Mol. Endocrinol.. 2009, 23: 1092-1101.

[35] Xie Jet al. . Transcript profiling of immediate early genes reveals a unique role for activating transcription factor 3 in mediating activation of the glycoprotein hormone alpha-subunit promoter by gonadotropin-releasing hormone. Mol. Endocrinol.. 2005, 19: 2624-2638.

[36] Liu F, Austin DA, Mellon PL, Olefsky JM, Webster NJ. GnRH activates ERK1/2 leading to the induction of c-fos and LHbeta protein expression in LbetaT2 cells. Mol. Endocrinol.. 2002, 16: 419-434

[37] Coss D, Jacobs SB, Bender CE, Mellon PL. A novel AP-1 site is critical for maximal induction of the follicle-stimulating hormone beta gene by gonadotropin-releasing hormone. J. Biol. Chem.. 2004, 279: 152-162.

[38] Ghosh MG, Thompson DA, Weigel RJ. PDZK1 and GREB1 are estrogen-regulated genes expressed in hormone-responsive breast cancer. Cancer Res.. 2000, 60: 6367-6375

[39] Sun J, Nawaz Z, Slingerland JM. Long-range activation of GREB1 by estrogen receptor via three distal consensus estrogen-responsive elements in breast cancer cells. Mol. Endocrinol.. 2007, 21: 2651-2662.

[40] Chen Cet al. . Optimized TrkB agonist ameliorates alzheimer’s disease pathologies and improves cognitive functions via inhibiting delta-secretase. ACS Chem. Neurosci.. 2021, 12: 2448-2461.

[41] Kang SS, Wu Z, Liu X, Edgington-Mitchell L, Ye K. Treating Parkinson’s disease via activation of BDNF/TrkB signaling pathways and inhibition of delta-secretase. Neurotherapeutics. 2022, 19: 1283-1297.

[42] Wang ZHet al. . Deficiency in BDNF/TrkB neurotrophic activity stimulates delta-secretase by upregulating C/EBPbeta in Alzheimer’s disease. Cell Rep.. 2019, 28: 655-669 e655.

[43] Zhang Z, Tian Y, Ye K. delta-secretase in neurodegenerative diseases: mechanisms, regulators and therapeutic opportunities. Transl. Neurodegener.. 2020, 9: 1.

[44] Qian Zet al. . Inhibition of asparagine endopeptidase (AEP) effectively treats sporadic Alzheimer’s disease in mice. Neuropsychopharmacology. 2024, 49620-630.

[45] Kondo T, Kitazawa R, Yamaguchi A, Kitazawa S. Dexamethasone promotes osteoclastogenesis by inhibiting osteoprotegerin through multiple levels. J. Cell Biochem.. 2008, 103: 335-345.

[46] Fu Q, Jilka RL, Manolagas SC, O’Brien CA. Parathyroid hormone stimulates receptor activator of NFkappa B ligand and inhibits osteoprotegerin expression via protein kinase A activation of cAMP-response element-binding protein. J. Biol. Chem.. 2002, 277: 48868-48875.

[47] Song Cet al. . Evaluation of efficacy on RANKL induced osteoclast from RAW264.7 cells. J. Cell Physiol.. 2019, 234: 11969-11975.

[48] Blick SK, Dhillon S, Keam SJ. Teriparatide: a review of its use in osteoporosis. Drugs. 2008, 68: 2709-2737.

[49] Lv Zet al. . Comparative study in estrogen-depleted mice identifies skeletal and osteocyte transcriptomic responses to abaloparatide and teriparatide. JCI Insight. 2023, 8: e161932.

[50] Ahn EHet al. . BDNF and Netrin-1 repression by C/EBPbeta in the gut triggers Parkinson’s disease pathologies, associated with constipation and motor dysfunctions. Prog. Neurobiol.. 2021, 198. 101905

[51] Bohaczuk, S. C., Thackray, V. G., Shen, J., Skowronska-Krawczyk, D. & Mellon, P. L. FSHB Transcription is Regulated by a Novel 5’ Distal enhancer with a fertility-associated single nucleotide polymorphism. Endocrinology162, bqaa181 (2021).

[52] Coss D, Hand CM, Yaphockun KK, Ely HA, Mellon PL. p38 mitogen-activated protein kinase is critical for synergistic induction of the FSH(beta) gene by gonadotropin-releasing hormone and activin through augmentation of c-Fos induction and Smad phosphorylation. Mol. Endocrinol.. 2007, 213071-3086.

[53] Wang Yet al. . Activator protein-1 and smad proteins synergistically regulate human follicle-stimulating hormone beta-promoter activity. Endocrinology. 2008, 149: 5577-5591.

[54] Simoni M, Gromoll J, Nieschlag E. The follicle-stimulating hormone receptor: biochemistry, molecular biology, physiology, and pathophysiology. Endocr. Rev.. 1997, 18: 739-773

[55] Huhtaniemi Iet al. . Genetically modified mouse models in studies of luteinising hormone action. Mol. Cell Endocrinol.. 2006, 252: 126-135.

[56] Layman LCet al. . Delayed puberty and hypogonadism caused by mutations in the follicle-stimulating hormone beta-subunit gene. N. Engl. J. Med.. 1997, 337: 607-611.

[57] Matthews CHet al. . Primary amenorrhoea and infertility due to a mutation in the beta-subunit of follicle-stimulating hormone. Nat. Genet.. 1993, 5: 83-86.

[58] Dattatreyamurty B, Figgs LW, Reichert LEJr. Physical and functional association of follitropin receptors with cholera toxin-sensitive guanine nucleotide-binding protein. J. Biol. Chem.. 1987, 262: 11737-11745.

[59] Lizneva, D. et al. FSH Beyond Fertility. Front. Endocrinol. 10, 136 (2019).

[60] Bhartiya D, Patel H. An overview of FSH-FSHR biology and explaining the existing conundrums. J. Ovarian Res.. 2021, 14. 144

[61] Gera, S. et al. FSH-blocking therapeutic for osteoporosis. Elife11, e78022 (2022).

[62] Fu SL, Pang H, Xu JZ, Wu XH. C/EBPbeta mediates osteoclast recruitment by regulating endothelial progenitor cell expression of SDF-1alpha. PLoS One. 2014, 9: e91217.

[63] Smink JJet al. . Transcription factor C/EBPbeta isoform ratio regulates osteoclastogenesis through MafB. EMBO J.. 2009, 281769-1781.

[64] Staiger Jet al. . C/EBPbeta regulates body composition, energy balance-related hormones and tumor growth. Carcinogenesis. 2009, 30: 832-840.

[65] Henriquez Bet al. . C/EBPβ binds the P1 promoter of the Runx2 gene and up-regulates Runx2 transcription in osteoblastic cells. J. Cell Physiol.. 2011, 226: 3043-3052.

[66] Smink JJ, Leutz A. Rapamycin and the transcription factor C/EBPbeta as a switch in osteoclast differentiation: implications for lytic bone diseases. J. Mol. Med.. 2010, 88: 227-233.

[67] Jafari Aet al. . Legumain regulates differentiation fate of human bone marrow stromal cells and is altered in postmenopausal osteoporosis. Stem Cell Rep.. 2017, 8: 373-386.

[68] Edgington-Mitchell LEet al. . Cysteine cathepsin activity suppresses osteoclastogenesis of myeloid-derived suppressor cells in breast cancer. Oncotarget. 2015, 6: 27008-27022.

[69] Bonaccorsi G, Piva I, Greco P, Cervellati C. Oxidative stress as a possible pathogenic cofactor of post-menopausal osteoporosis: existing evidence in support of the axis oestrogen deficiency-redox imbalance-bone loss. Indian J. Med. Res.. 2018, 147: 341-351.

[70] Liang, G. et al. Menopause-associated depression: impact of oxidative stress and neuroinflammation on the central nervous system-a review. Biomedicines12, 184 (2024).

[71] Mohamad NV, Ima-Nirwana S, Chin KY. Are oxidative stress and inflammation mediators of bone loss due to estrogen deficiency? A review of current evidence. Endocr. Metab. Immune Disord. Drug Targets. 2020, 20: 1478-1487.

[72] Pulido-Salgado M, Vidal-Taboada JM, Saura J. C/EBPbeta and C/EBPdelta transcription factors: basic biology and roles in the CNS. Prog. Neurobiol.. 2015, 132: 1-33.

[73] van der Krieken SE, Popeijus HE, Mensink RP, Plat J. CCAAT/enhancer binding protein beta in relation to ER stress, inflammation, and metabolic disturbances. Biomed. Res. Int.. 2015, 2015324815

[74] Shirahama-Noda Ket al. . Biosynthetic processing of cathepsins and lysosomal degradation are abolished in asparaginyl endopeptidase-deficient mice. J. Biol. Chem.. 2003, 278: 33194-33199.

[75] Arlt Het al. . Effects of abaloparatide and teriparatide on bone resorption and bone formation in female mice. Bone Rep.. 2020, 13. 100291

[76] Weiss J, Guendner MJ, Halvorson LM, Jameson JL. Transcriptional activation of the follicle-stimulating hormone beta-subunit gene by activin. Endocrinology. 1995, 136: 1885-1891.

[77] Coss D, Thackray VG, Deng CX, Mellon PL. Activin regulates luteinizing hormone beta-subunit gene expression through Smad-binding and homeobox elements. Mol. Endocrinol.. 2005, 19: 2610-2623.

[78] Chevalier Cet al. . Primary mouse osteoblast and osteoclast culturing and analysis. STAR Protoc.. 2021, 2. 100452

[79] Li JYet al. . IL-17A is increased in humans with primary hyperparathyroidism and mediates pth-induced bone loss in mice. Cell Metab.. 2015, 22: 799-810.

[80] Li JYet al. . Sex steroid deficiency-associated bone loss is microbiota dependent and prevented by probiotics. J. Clin. Invest.. 2016, 126: 2049-2063.

[81] Dempster DWet al. . Standardized nomenclature, symbols, and units for bone histomorphometry: a 2012 update of the report of the ASBMR Histomorphometry Nomenclature Committee. J. Bone Miner. Res.. 2013, 28: 2-17.

Funding

National Natural Science Foundation of China (National Science Foundation of China)(32330040)

Shenzhen Science and Technology Innovation Commission(KQTD2022110 1093608028)

National Science Foundation of China | Young Scientists Fund(82501723)

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The Author(s)