obesity increases the risk of developing cardiovascular disease , cancer , and type 2 diabetes . despite their adiposity , some obese individuals do not develop these diseases and otherwise retain good metabolic health [ 13 ] . this suggests that impairments in adipose tissue ( at ) function drive metabolic health independently of body adiposity . two such impairments , adipose tissue fibrosis and inflammation , are tightly associated with insulin resistance and the metabolic syndrome ( mets ) [ 46 ] , while the transcription factor hypoxia inducible factor 1 ( hif-1 ) induces fibrosis in ats . additionally , sex hormones and their receptors attenuate at fibrosis and inflammation [ 913 ] . in this report we present evidence linking the metabolically protective effect of estrogens and estrogen receptors with hif-1 activity in at . in conditions of caloric excess , adipose tissue expands to accommodate increased lipid stores . if the expansion outpaces the growth of the vasculature , hypoxia in the at depot develops [ 1418 ] . hypoxia provokes a transcriptional response regulated by hif-1 , a heterodimeric transcription factor consisting of two subunits , hif-1 and hif-1. cells constitutively express both subunits ; however , hif-1 is highly sensitive to post - translational degradation . in normoxic conditions , three isoforms of prolyl-4-hydroxylases ( phds ) hydroxylate hif-1 in an oxygen - dependent manner , targeting it for ubiquitination by the von hippel ubiquitinated hif-1 is ultimately degraded by the proteasome . under hypoxia , phds fail to catalyze hif-1 hydroxylation due to lack of adequate oxygen . stabilized hif-1 translocates to the nucleus and binds hif response elements ( hres ) in proangiogenic and metabolism related genes in a wide variety of tissues . , hif-1 promotes transcription of pro - fibrotic and inflammatory gene targets , including lysyl oxidase ( lox ) and il6 
 . fibrosis and inflammation , in turn , induce systemic insulin resistance . genetic and pharmacological inhibition of hif-1 in obese mice reduces at fibrosis and improves insulin resistance independent of adiposity , implicating hif-1 as a mediator of metabolically  unhealthy  at . it is established that adipose tissue fibrosis and inflammation lead to insulin resistance , mets and type 2 diabetes ( t2 dm ) . for example , women generally possess a higher total body fat percentage than men , but men tend to accrue a higher percentage of visceral adipose tissue ( vat ) , which increases the risk of mets and cardiovascular disease . premenopausal women are protected from t2 dm and heart disease but lose this protection with increasing visceral adiposity after menopause . the metabolic protection afforded by sex can be partially attributed to the actions of estrogens and estrogen receptors ( ers ) . estrogens function as ligands for estrogen receptor  ( er ) , estrogen receptor  ( er ) , and the membrane bound g - protein - coupled estrogen receptor ( gper ) . once bound , the estrogenic complex recognizes estrogen response elements ( eres ) on genomic and mitochondrial dna and promotes transcription of genes related to proliferation , differentiation and nutrient uptake . for example , post - menopausal women experience reduced 17- estradiol ( e2 ) levels and a corresponding increase of vat , which is associated with increased risk of t2 dm . ovariectomy ( ovx ) with concomitant reductions in e2 in mice induces at fibrosis , inflammation and insulin resistance . similarly , both global knockout of er ( erko ) in mice and tissue specific knockdown of er in adipocytes in  vivo induces fibrosis and insulin resistance , demonstrating er mediates e2 's actions on fibrosis and inflammation in at . both er and hif-1 are related to fibrosis and inflammation in adipose tissue , albeit in opposite ways : while er improves at function , hif-1 worsens it . here , we demonstrate e2/er regulates hif-1 activity in at by promoting transcription of a specific phd , thus providing a mechanistic explanation for the protective effect of e2/er against the metabolic impact of hif-1 activation in at . mice were housed in cages with a 12-h light / dark cycle and with ad libitum access to water and standard chow ( # 2916 , harlan - teklad ) . all animal experimental protocols were approved by the institutional animal care and use committee of university of texas southwestern medical center at dallas . adipose tissues were collected for immunostaining at 95 days of age ; otherwise , mice were 11 or 12 weeks old at time of experiments . 293 t , 3t3-l1 cells , nih3t3 were obtained from atcc and cultured as follows : 3t3-l1 cells were grown to confluence dulbecco 's modified eagle 's medium ( dmem , life technologies ) supplemented with 10% calf serum and antibiotics ( penicillin and streptomycin ) . differentiation into adipocytes was induced by exposure to differentiation media ( dmem 10% fetal bovine serum ( fbs ) , 0.5  mm 3-isobutyl-1-methylxanthin ( ibmx ) , 0.25  m dexamethasone and 5  g / ml insulin ) for 48  h. thereafter , adipocytes were cultured in maintenance media ( dmem supplemented with 10% fbs and 1  g / ml insulin ) changed every 2 days . at day 8 of differentiation , adipocytes were used for experiments . dissected fat tissue from each mouse was washed , minced and then digested for 45  min  at 37  c in digestion buffer ( 100  mm hepes ( ph 7.4 ) , 120  mm nacl , 50  mm kcl , 5  mm glucose , 1  mm cacl2 , 1  mg / ml collagenase d ( roche ) , and 1.5% bsa ) . digested tissue was then diluted with svf medium ( high glucose dmem / f12  +  glutamax ( invitrogen ) , 10% fbs ) and filtered through a 210  m cell strainer to remove undigested tissues . the flow - through was then diluted with svf medium and centrifuged for 5  min  at 600    g to pellet the stromal - vascular ( sv ) cells and separate the adipocytes . floated adipocytes were collected and sv cells were re - suspended in svf medium and filtered through a 40  m cell strainer to remove clumps and large adipocytes . following a second centrifugation , sv cells were homogenized in trizol ( invitrogen ) for rna extraction . to generate the mouse hif-1 expression vector , philip shaul ( ut southwestern medical center , dallas , tx ) . hif response element ( hre)-luciferase vector was obtained courtesy of dr . richard bruick ( ut southwestern medical center , dallas , tx ) and myc - ub1 and pcmv-gal vectors were obtained courtesy of kyong soo park ( seoul national university , korea ) . estrogen response element ( ere)-luciferase was constructed as follows : dna fragments containing from 2077 , 680 to 122  bp from transcription start site of the mouse phd3 gene were cloned into the region upstream of the luciferase gene of the pgl3-basic vector ( promega ) to generate phd3 reporter candidates ( referred to as candidates 2038 , 1832 , 1005 , 680 ) . lipofectamin ltx ( invitrogen ) and plus reagents were used for transfection of all plasmids . adenoviruses overexpressing flag - er ( ad - er ) , phd3 ( ad - phd3 ) and gfp ( ad - gfp ) were generated as described previously . briefly , n - terminal flag - tagged her , and mouse phd3 fragments were inserted into padtrack - cmv shuttle vector . for sher ( 5-ggacuugaaucuccaugau-3 ) and shphd3 ( 5-ggagccggcugggcaaaua-3 ) adenovirus , we used prnat h1.1-adeno vector . intact shuttle vector of padtrack - cmv or negative control sequence inserted prnat h1.1-adeno vector ( genescript ) were used for construction of control virus ( ad - gfp and adns : 5-gccuacgccaccaauuucgu-3 ) . cloned shuttle vectors were linearized with pmei and then subjected to electrophoration for homologous recombination between shuttles and adeasy-1 viral vector in adeasier ( stratagene ) electrocompetent cells . adenoviral vectors were linearized with paci and were amplified using ad 293 cells ( stratagene ) . 109  pfu of adenoviral particles were injected into mouse inguinal fat pads . in each experiment , the selection of which fat pad received the expression / interference or control vector was randomized . the specifics of each injection experiment are as follows : for er overexpression , 11-week - old female wt c57/bl6 ( n  =  9 ) and female erko mice ( n  =  11 ) were injected with er overexpression vector ( ad - er ) in one inguinal at pad and a control vector ( ad - gfp ) in the contra - lateral pad.for er interference , 11-week - old female wt c57/bl6 mice ( n  =  9 ) were injected with er interference vector ( sher ) in one inguinal at pad and a control vector ( shns ) in the contra - lateral pad.for phd3 overexpression , 12-week - old female c57/bl6 ( n  =  7 ) and female erko mice ( n  =  8) were injected with phd3 overexpression vector ( ad - phd3 ) in one inguinal at pad and a control vector ( ad - gfp ) in the contra - lateral pad.for phd3 interference , 12-week - old female c57/bl6 mice ( n  =  7 ) were injected with phd3 interference vector ( shphd3 ) in one inguinal fat pad and a control vector ( shns ) in the contra - lateral pad . for er overexpression , 11-week - old female wt c57/bl6 ( n  =  9 ) and female erko mice ( n  =  11 ) were injected with er overexpression vector ( ad - er ) in one inguinal at pad and a control vector ( ad - gfp ) in the contra - lateral pad . n  =  9 ) were injected with er interference vector ( sher ) in one inguinal at pad and a control vector ( shns ) in the contra - lateral pad . for phd3 overexpression , 12-week - old female c57/bl6 ( n  =  7 ) and female erko mice ( n  =  8) were injected with phd3 overexpression vector ( ad - phd3 ) in one inguinal at pad and a control vector ( ad - gfp ) in the contra - lateral pad . for phd3 interference , 12-week - old female c57/bl6 mice ( n  =  7 ) were injected with phd3 interference vector ( shphd3 ) in one inguinal fat pad and a control vector ( shns ) in the contra - lateral pad . for hre luciferase assay , nih3t3 cells were plated in 24-well culture plates and transfected with 300  ng of hre luc and 20  ng pcmv-gal and with or without 100  ng of pcdna - her. cells were then incubated with or without e2 ( 10  nm ) for 48  h  in phenol red - free dmem supplemented with 2% fbs . two days after transfection , cells were harvested using lysis buffer and luciferase activity was determined according to manufacturer 's instruction ( promega ) . hre luciferase activities were normalized to the activity of hre luc in the absence of er expression . for phd3 promoter ere luciferase assay , 293 t cells were plated in 24-well culture plates and transfected with 300  ng of ere luc and 20  ng pcmv-gal with or without 200  ng sher. cells were then incubated with either e2 ( 10  nm ) or propyl pyrozole triol ( ppt ) ( 10  nm ) ( cayman chemical ) . after 48  h  cells were harvested using lysis buffer and luciferase activity was determined according to manufacturer 's instruction ( promega ) . ere luciferase activities were normalized to the activity of phd3 ( 2077 ) luc in the absence of er expression , e2 or ppt . chip was carried out in differentiated 3t3-l1 adipocytes or nih3t3 cells using the ez - chip chromatin immunoprecipitation kit ( millipore ) according to manufacturer 's instructions . briefly , cells were treated with either 0 or 10  nm e2 or ppt ( 10  nm ) for 18  h. cells were then treated with 1% formaldehyde to induce protein chromatin cross - linking ; 0.125  m glycine was used to quench unreacted formaldehyde . chromatin fragments were immunoprecipitated with either er antibody ( santa cruz , santa cruz , ca , er mc-20 , # sc-542 ) or igg ( santa cruz ) and selected with protein g sepharose ( millipore ) . after reverse cross - linking , dna was purified for detection using pcr . pcr primers used delimit four candidate eres in the phd3 promoter and are listed in supplementary table  1 . nih3t3 cells were plated in 6-cm dishes and transfected with pcdna - mhif1 , myc - ub1 and pflag - er , shphd3 plasmids . after incubation for 48  h , cell lysates were obtained with lysis buffer ( 20  mm tris ( ph7.4 ) , 1% np40 , 5  mm edta , 10  mm na4p2o7 , 100  mm naf , 2  mm na3vo4 , 5  g / ml aprotinin , 5  g / mg leupeptin , 1  mm pmsf ) . lysates were briefly sonicated and then incubated with 2  g of hif1 antibody ( novus , littleton , co , nb100 - 449 ) for 6  h and protein g sepharose ( ge healthcare ) for another 2  h. washed precipitates were immunoblotted with anti - myc antibody ( sigma ) . collected inguinal ats the protein bands were detected with species - specific secondary antibodies ( santa cruz ) conjugated with infrared dyes and visualized with li - cor odyssey infrared scanner ( li - cor bioscience ) . adipose tissues were collected from 95 day old mice and fixed overnight in 10% formalin . the molecular pathology core at ut southwestern medical center embedded tissues with paraffin , sectioned and immunostained with phd3 antibody ( novus , nb100 - 303 ) or er antibody ( santa cruz , er mc-20 , # sc-542 ) . adipose tissues were excised and homogenized in trizol using a tissuelyser lt ( qiagen ) and steel beads . for both cell culture and adipose tissue , total rnas were extracted in trizol ( invitrogen ) and isolated using the rneasy rna extraction kit ( qiagen ) according to the manufacturer 's instructions . the quantity and quality of the rna were determined by absorbance at 260/280  nm . cdna was prepared by reverse transcribing 1  g rna with superscript iii reverse transcriptase ( invitrogen ) and oligo(dt ) ( invitrogen ) . quantitative real - time pcrs ( qpcrs ) were performed with taqman or sybr gene - specific primers on an abi prism 7900 ht sequence detection system ( applied biosystems ) using technical duplicates . the relative amounts of all mrnas were calculated by using the comparative threshold cycle ( ct ) method . glyceraldehyde-3-phosphate dehydrogenase ( gapdh ) , -2 microglobulin ( b2 m ) or hypoxanthine guanine phosphoribosyl transferase ( hprt ) mrna were used as the endogenous controls . all the results are presented as means    standard error of the mean ( s.e.m . ) . statistical significance between groups was determined by two - tailed student 's t - test or one - way anova . initially we demonstrated that there is a sexual dimorphism in er protein and message in adipose tissue , with males having significant reductions in er relative to females ( figure  1b and c ) . we hypothesized that reduction of er in ats promotes hif-1 transcriptional activity . to evaluate this effect addition of both er and e2 significantly decreased hif-1 promoter driven luciferase activity ( figure  1a ) , whereas the addition of er alone did not have an effect , suggesting ligand dependence . e2 and ers positively regulate expression of phd1 and phd2 in breast cancer and epithelial cells , respectively . since phds are critical regulators of hif-1 activity , we examined how e2/er influences phds expression in perigonadal at . e2/er positively regulated phd3 but not phd1 or phd2 mrna in perigonadal at of lean , weight matched , intact / cycling female wt , male wt and female er knockout ( erko ) mice ( figure  1d ) . to determine if a functional relationship exists between er , phd3 and hif-1 activity , we inhibited phd3 using rna interference . knockdown of phd3 abrogated the effect of e2/er on hif-1 promoter driven luciferase , demonstrating that phd3 is required for reduction of hif-1 activity ( figure  1a ) . adipocytes represent only a fraction of the overall cell population of ats . to determine if phd3 is expressed specifically in adipocytes , we separated whole at into adipocyte - rich and adipocyte - poor fractions ( svf ) . the adipocyte - rich fraction strongly expressed adiponectin , an adipocyte - specific adipokine , while the adipocyte - poor fraction did not , confirming a clear separation by cell type ( supplementary figure  1 ) . female wt adipocytes expressed significantly more phd3 mrna than either female erko or male wt ( figure  1e ) . immunohistochemistry for phd3 in perigonadal at verified the up - regulation of protein levels in adipocytes ( figure  1f ) , further demonstrating that females have higher levels of phd3 than males or erko mice . next , we utilized chromatin immunoprecipitation ( chip ) and luciferase reporter assays to determine whether er directly activates phd3 gene expression . computational analysis revealed potential eres in the proximal promoter region of phd3 ( figure  2a ) . location analysis by chip - qpcr revealed that er binds to an ere 599  bp upstream of the phd3 promoter ( candidate 599 ) in differentiated 3t3-l1 adipocytes , but not at other potential eres ( figure  2b , supplementary table  1 ) . treatment with e2 or ppt , an er-specific agonist , significantly increased erere binding ( figure  2b ) , indicating a ligand - dependent effect . in luciferase reporter assays where the phd3 promoter drives luciferase expression , only constructs including candidate 599 responded significantly to e2/er , further indicating a ligand - dependent effect on phd3 transcription ( figure  2c , supplementary table  1 ) . in order to determine if the erphd3 regulatory axis leads to hif-1 ubiquitination , we blocked proteosomal activity with mg132 and measured ubiquitinated hif-1 in the presence and absence of er. addition of er increased hif-1 ubiquitination ; this effect depended on phd3 ( figure  2d , supplementary figure  2b ) . to assess the effect of er on phd3 expression in  vivo , we injected the inguinal ats of wt and erko mice with er overexpressing or silencing vectors ( supplementary figure  3 ) . importantly , all of these experiments were done in intact female mice , without manipulation of the hormonal milieu . at baseline , erko ats expressed less phd3 mrna than wt ( figure  3b ) . additionally , erko ats had higher mrna levels of lox , a direct transcriptional target of hif-1 , as well as markers of inflammation ( figure  3c and d ) . in erko mice , the er overexpression vector increased er expression ( figure  3a ) . restoration of er directly in at increased expression of phd3 , but not phd1 or phd2 mrnas ( figure  3b ) , and reduced expression of inflammatory genes , as well as lox , to wt levels ( figure  3c and d ) . in female wt mice , knockdown of er produced the opposite effect : phd3 expression decreased ( figure  4a ) , while expression of lox and inflammation markers significantly increased ( figure  4b and c ) . dysfunctional at is characterized by increased fibrosis and inflammation . to demonstrate the ability of phd3 to ameliorate fibrosis and inflammation in at , we performed phd3 gain and loss of function experiments in  vivo as previously described . addition of phd3 to erko at significantly reduced expression of col11 and col31 mrnas ( figure  5a and c ) . consistent with a reduction in hif-1 activity , addition of phd3 decreased lox and il6 mrnas ( figure  6b and c ) . knockdown of phd3 in wt mice did not otherwise affect er levels ( figure  6a ) and induced markers of fibrosis and inflammation , particularly lox and col11 and col31 , as well as tnf and tlr-4 mrnas ( figure  6b and c ) . e2 and ers protect against inflammation and insulin resistance in at ; however , the mechanism underlying this phenomenon is unknown . activity produces fibrosis , inflammation and insulin resistance in at [ 68 ] . in this report we link e2/er and hif-1 , demonstrating that e2/er directly promotes transcription of phd3 , a key enzyme in hif-1 regulation , thus reducing markers of fibrosis and inflammation . the discovery of an e2/er dependent mechanism of hif-1 regulation contributes to our understanding of the sexual dimorphism evident in at function . previous reports describe an association between e2/er and hif-1 protein levels . for example , removal of ovarian estrogens by ovx in rats results in hypoxic visceral at and increased hif-1 activity , while er overexpression in mcf-7 cells down - regulates hif-1 activity . consistent with these studies , we demonstrate that e2/er reduces hif-1 transcriptional activity in  vitro and in an at specific manner in  vivo . recently , miyauchi et  al . showed that e2 treatment destabilizes hif-1 in osteoclasts in  vitro , and that osteoclast specific deletion of er stabilizes hif-1 in  vivo 
 . interestingly , that study found no evidence of increased ubiquitination or proteosomal degradation of hif-1 , concluding that the regulatory mechanism was not related to the classical hif-1 ubiquitination pathway . however , we found that e2/er increases ubiquitination of hif-1 , with consequent reduction of hif-1 activity in at . our data extends the association of e2/er with hif-1 to include a heretofore uncharacterized interaction with prolyl-4-hydroxylase enzymes ( phds ) . the phd family comprises three structurally similar and functionally overlapping enzymes , two of which have been associated with er activity in tissues other than at . e2 positively regulates phd1 in breast cancer cells , while er promotes phd2 transcription in epithelial cells . therefore , we hypothesized that e2/er may affect hif-1 activity via regulation of one of the phd genes . we found interference with phd3 induces hif-1 activity ( figure  1a ) ; neither phd1 nor phd2 were regulated by sex , estrogens , or er in our models ( figure  1d ) . examination of erko and wt tissues revealed that phd3 mirrors er levels in ats and , specifically , in adipocytes : female wt ats are enriched in e2/er and phd3 compared to males ( which have substantially lower e2/er ) and erko ( which lack er ) ats ( figure  1b  f ) . moreover , selective knockdown of er in the inguinal at of wt mice proves that the disparity in phd3 depends on er and is independent of body adiposity ( figure  4 ) . importantly , loss of er induces lox , a known hif-1 target , showing that hif-1 protein is , indeed , transcriptionally active in these cells under these conditions ( figure  4b ) . as such , it is not unreasonable to suspect that er could promote phd3 transcription . however , in erko mice , which have er , we observed reduced phd3 expression , indicating that er does not influence this pathway appreciably ( supplementary figure  4a  d ) . this does not , however , completely rule out a modulatory role for er in mediating phd3 transcription . we observed that er only reduced hre luciferase significantly in the presence of e2 ( figure  1a ) , suggesting that the erphd3 pathway is ligand - dependent . in our subsequent chip and ere luciferase experiments , e2 and the er agonist ppt significantly induce ere binding as well as promoter driven transcription . since ppt is an er specific agonist , this is consistent with conventional mechanisms of er gene transcription   and with the observation that decrements in at function and health accompany declining levels of ovarian estrogens in post - menopausal women . studies have concluded that phd3 preferentially hydroxylates hif-2 rather than hif-1 and only becomes a significant contributor to hif-1 regulation in hypoxic conditions . our results do not refute these findings ; rather , taken with our identification of an ere in the phd3 promoter and findings that er and phd3 are predominately expressed in the adipocyte fraction of adipose tissue , they demonstrate a novel regulatory relationship between e2/er and phd3 in adipocytes . while obesity - induced adipose tissue hypoxia has been implicated as the root cause of hif-1 activation and consequent metabolic dysfunction , the transcriptional program driven by hif-1 in at requires neither obesity nor hypoxia . for example , in transgenic models , hif-1 overexpression in normoxia led to similar increases in fibrosis , inflammation and insulin resistance as observed in obese , hypoxia models . we have also recently reported that fibrosis and inflammation characterize the at of both lean and obese erko mice as well as in mice with a selective knockdown of er from adipocytes . therefore , we determined if in  vivo manipulation of the erphd3 transcriptional mechanism could affect hif-1 activity to induce similar markers of fibrosis and inflammation in lean mice . importantly , inhibition of phd3 in lean , wt mice promotes lox and collagen expression , as well as tnf and tlr-4 expression , demonstrating that phd3 alone can protect from at fibrosis and inflammation ( figure  6b and c ) . to further demonstrate the critical roles of e2/er in regulating adipose tissue inflammation and fibrosis , we determined that restoration of er in erko ( which have elevated circulating levels of e2 ) reduce both lox and tnf ( figure  3b and c ) , and that restoration of phd3 in erko also rescues the at phenotype ( figure  5b and c ) . in addition to serving as a proxy for hif-1 activity , lox induction has important metabolic consequences in at . lox is a hif-1 sensitive oxidase that crosslinks fibrils and insolubilizes components of the ecm . previous reports implicate lox in ecm remodeling and inflammation , leading to impaired insulin sensitivity and glucose intolerance in at . indeed , inhibition of lox reversed approximately 900 genes identified by microarray as dysregulated in hif-1 overexpressing transgenic mice . in our model , we observed greater expression of lox in erko adipose at baseline , while restoration of er or phd3 was sufficient to reverse both lox and inflammatory markers ( figures  3 and 5 ) . loss of at er or phd3 in wt female mice replicated these findings , further evidence that the erphd3 mechanism operates in a physiologically relevant way ( figures  4 and 6 ) . the anti - fibrotic effect of er restoration was more pronounced than the pro - fibrotic effect of er knockdown . we attribute this to the fact er was restored in erko mice , which are known to have elevated levels of e2 , and the presence of ligand strongly induces the erphd3 transcriptional mechanism . conversely , knockdown of er in the wt mice does not result in complete removal of er from at . the erphd3 pathway is weakened , but not completely absent , leading to a modest elevation of hif-1 induced fibrosis . while hif-1 has been studied extensively in the context of cancer and hypoxic tumor growth , its contributions to normoxic metabolism are less clear . indeed , precise calculations of the level of oxygenation required to attenuate hif-1 activity are technically difficult and inconsistently reported in the literature . presently , it is unclear if e2/er promotes a more robust response to chronic at hypoxia , thus mitigating the deleterious effects of hif-1 activity in obesity , or if higher basal expression of phd3 in er-rich ats protects against such activity . evidence suggests that phd3 is more strongly induced , as well as retains its catalytic ability under prolonged hypoxia better than the other phd isoforms . under non - reversible reaction conditions , it is reasonable to hypothesize that adipocytes expressing er will accumulate more phd3 , thereby reducing hif-1 driven fibrosis and inflammation . accordingly , investigations using hypoxia or hypoxia mimetics should be pursued to more precisely define the contribution of er induced phd3 expression to the hypoxic response . in addition to hif-1 regulation , phd3 has been linked to several important cellular pathways . loss of phd3 has been shown to decrease cell adhesion proteins , increasing the metastatic potential of tumors , while expression of phd3 correlates with favorable tumor prognosis . phd3 has also been implicated in the regulation of apoptosis in a variety of cell lines . the transcriptional relationship between e2/er and phd3 sheds light on these and other phd - related pathways . furthermore , demonstration that phd3 inhibition is pro - fibrotic and pro - inflammatory in at has important clinical implications . recent reports suggest that phd3 inhibition improves liver insulin sensitivity , while clinical studies are currently underway to assess the efficacy of phd inhibition in the treatment of a variety of pathologies , including anemia and peripheral artery disease . since fibrosis and inflammation have been causally linked to insulin insensitivity , mets and t2 dm , our studies indicate that loss of phd3 functionality in at could have negative metabolic consequences . the effect of phd inhibition interventions on at function must be taken into account , especially for long - term treatments . here we demonstrate that e2/er is a direct transcriptional regulator of phd3 , a key regulatory enzyme in the hif-1 degradation pathway . thus , ats are protected from hif-1 induced fibrosis and inflammation by an e2/er dependent , phd3 mediated mechanism , as depicted in the cartoon summarizing the proposed mechanism ( figure  7 ) . the revelation that e2/er participates in a direct regulatory pathway with phd3 and hif-1 provides a mechanistic explanation for the protective effect of e2/er regarding at function .
hypoxia inducible factor 1 ( hif-1 ) promotes fibrosis and inflammation in adipose tissues , while estrogens and estrogen receptor  ( er ) have the opposite effect . here 
 we identify an estrogen response element ( ere ) in the promoter of phd3 , which is a negative regulatory enzyme of hif-1 , and we demonstrate hif-1 is ubiquitinated following 17- estradiol ( e2)/er mediated phd3 transcription . manipulating er in  vivo increases phd3 transcription and reduces hif-1 activity , while addition of phd3 ameliorates adipose tissue fibrosis and inflammation . 
 our findings outline a novel regulatory relationship between e2/er , phd3 and hif-1 in adipose tissues , providing a mechanistic explanation for the protective effect of e2/er in adipose tissue .