Target Deletion of the Cytoskeleton-Associated Protein Palladin Does Not Impair Neurite Outgrowth in Mice

Palladin is an actin cytoskeleton–associated protein which is crucial for cell morphogenesis and motility. Previous studies have shown that palladin is localized to the axonal growth cone in neurons and may play an important role in axonal extension. Previously, we have generated palladin knockout mice which display cranial neural tube closure defect and embryonic lethality before embryonic day 15.5 (E15.5). To further study the role of palladin in the developing nervous system, we examined the innervation of palladin-deficient mouse embryos since the 200 kd, 140 kd, 90–92 kd and 50 kd palladin isoforms were undetectable in the mutant mouse embryo brain. Contrary to the results of previous studies, we found no inhibition of the axonal extension in palladin-deficient mouse embryos. The cortical neurons derived from palladin-deficient mice also showed no significant difference in neurite outgrowth as compared with those from wild-type mice. Moreover, no difference was found in neurite outgrowth of neural stem cell derived-neurons between palladin-deficient mice and wild-type mice. In conclusion, these results suggest that palladin is dispensable for normal neurite outgrowth in mice.     

Redox Regulation of the Human Dual Specificity Phosphatase YVH1 through Disulfide Bond Formation

YVH1 was one of the first eukaryotic dual specificity phosphatases cloned, and orthologues posses a unique C-terminal zinc-coordinating domain in addition to a cysteine-based phosphatase domain. Our recent results revealed that human YVH1 (hYVH1) protects cells from oxidative stress. This function requires phosphatase activity and the zinc binding domain. This current study provides evidence that the thiol-rich zinc-coordinating domain may act as a redox sensor to impede the active site cysteine from inactivating oxidation. Furthermore, using differential thiol labeling and mass spectrometry, it was determined that hYVH1 forms intramolecular disulfide bonds at the catalytic cleft as well as within the zinc binding domain to avoid irreversible inactivation during severe oxidative stress. Importantly, zinc ejection is readily reversible and required for hYVH1 activity upon returning to favorable conditions. This inimitable mechanism provides a means for hYVH1 to remain functionally responsive for protecting cells during oxidative stimuli.Human YVH1 (hYVH1²; also known as DUSP12) is a member of the dual specificity phosphatase (DUSP) subfamily of protein-tyrosine phosphatases (PTPs) (1, 2). It is constructed of an N-terminal DUSP catalytic domain and a unique C-terminal zinc coordinating domain (3). Poor characterization and lack of mitogen-activated protein kinase targeting motifs further classify this enzyme as an atypical DUSP (1). YVH1 orthologues exhibit high evolutionary conservation and similar domain organization (3). Deletion of the yvh1 gene in yeast disrupts normal growth processes (4), whereas insertion and expression of the hyvh1 gene is capable of restoring a normal yeast growth phenotype (3). Amplification of the dusp12/hyvh1 gene has been reported in multiple sarcomas, implicating a role for hYVH1 in human disease (5–7).Recently, deletion studies from our laboratory have shown that the C-terminal zinc binding domain of hYVH1 is not essential for intrinsic phosphatase activity in vitro; however, it is required for interaction with the ATPase domain of heat shock protein 70 (8). Similarly, overexpression of wild type hYVH1 but not catalytically dead or zinc coordinating domain deletion mutants prevents cell death induced by Fas receptor activation, heat shock, and hydrogen peroxide (H₂O₂) (8). Despite these findings, current information on hYVH1 enzymatic and physiological functions remains limited.PTPs and DUSPs share similar active site architecture and catalytic mechanism, characterized by the conserved HCX₅R(S/T) motif (9, 10). The unique microenvironment within the HCX₅R(S/T) motif reduces the pK_a value of the active site cysteine, enhancing both nucleophilicity and oxidation susceptibility (11, 12). Stimulated or constituent generation of ROS can result in oxidative second messenger signaling responses capable of transient and reversible post-translational inactivation of both PTPs and DUSPs through oxidation of the catalytic cysteine (13–15).This oxidative susceptibility and modification varies among PTPs and DUSPs, a likely consequence of slight variations in active site conformations or mediated through unique regulatory domains (16–18). Accumulating evidence suggests that redox-mediated oxidation of PTPs is a dynamic modification that can differentially regulate PTPs (13, 19). Sulfenic acid, cyclic sulfenamide, and disulfide bond formation have all been shown to facilitate stable, reversible active site modifications among various PTPs and DUSPs (12, 14, 20). Furthermore, evidence suggests that oxidation predominantly and rapidly targets the active site cysteine, whereas other cysteinyl residues remain in the reduced state (15, 20).This study investigated the relationship between the zinc-coordinating C-terminal domain and the catalytic domain of hYVH1 during oxidative conditions. We provide data suggesting that the zinc binding domain can serve as a reducing agent during oxidative stress to impede the oxidation of the active site cysteine. Increased exposure to oxidative conditions readily induces disulfide bond formation within the zinc-coordinating and catalytic domains, resulting in concomitant zinc ejection and enzymatic inactivation. Zinc ejection is readily reversible and required for hYVH1 activity upon returning to reducing conditions. Thus, we propose a mechanism for phosphatase active site protection through the intrinsic redox buffering capacity of this unique zinc binding domain.     

Specificity Profiling of Dual Specificity Phosphatase Vaccinia VH1-related (VHR) Reveals Two Distinct Substrate Binding Modes

Vaccinia VH1-related (VHR) is a dual specificity phosphatase that consists of only a single catalytic domain. Although several protein substrates have been identified for VHR, the elements that control the in vivo substrate specificity of this enzyme remain unclear. In this work, the in vitro substrate specificity of VHR was systematically profiled by screening combinatorial peptide libraries. VHR exhibits more stringent substrate specificity than classical protein-tyrosine phosphatases and recognizes two distinct classes of Tyr(P) peptides. The class I substrates are similar to the Tyr(P) motifs derived from the VHR protein substrates, having sequences of (D/E/φ)(D/S/N/T/E)(P/I/M/S/A/V)pY(G/A/S/Q) or (D/E/φ)(T/S)(D/E)pY(G/A/S/Q) (where φ is a hydrophobic amino acid and pY is phosphotyrosine). The class II substrates have the consensus sequence of (V/A)P(I/L/M/V/F)X_1–6pY (where X is any amino acid) with V/A preferably at the N terminus of the peptide. Site-directed mutagenesis and molecular modeling studies suggest that the class II peptides bind to VHR in an opposite orientation relative to the canonical binding mode of the class I substrates. In this alternative binding mode, the Tyr(P) side chain binds to the active site pocket, but the N terminus of the peptide interacts with the carboxylate side chain of Asp¹⁶⁴, which normally interacts with the Tyr(P) + 3 residue of a class I substrate. Proteins containing the class II motifs are efficient VHR substrates in vitro, suggesting that VHR may act on a novel class of yet unidentified Tyr(P) proteins in vivo.     

Acid Suppression Therapy Does Not Predispose to Clostridium difficile Infection: The Case of the Potential Bias

Objective

An adverse effect of acid-suppression medications on the occurrence of Clostridium difficile infection (CDI) has been a common finding of many, but not all studies. We hypothesized that association between acid-suppression medications and CDI is due to the residual confounding in comparison between patients with infection to those without, predominantly from non-tested and less sick subjects. We aimed to evaluate the effect of acid suppression therapy on incidence of CDI by comparing patients with CDI to two control groups: not tested patients and patients suspected of having CDI, but with a negative test.

Methods

We conducted a case-control study of adult patients hospitalized in internal medicine department of tertiary teaching hospital between 2005–2010 for at least three days. Controls from each of two groups (negative for CDI and non-tested) were individually matched (1∶1) to cases by primary diagnosis, Charlson comorbidity index, year of hospitalization and gender. Primary outcomes were diagnoses of International Classification of Diseases (ICD-9)–coded CDI occurring 72 hours or more after admission.

Results

Patients with CDI were similar to controls with a negative test, while controls without CDI testing had lower clinical severity. In multivariable analysis, treatment by acid suppression medications was associated with CDI compared to those who were not tested (OR = 1.88, p-value = 0.032). Conversely, use of acid suppression medications in those who tested negative for the infection was not associated with CDI risk as compared to the cases (OR = 0.66; p = 0.059).

Conclusions

These findings suggest that the reported epidemiologic associations between use of acid suppression medications and CDI risk may be spurious. The control group choice has an important impact on the results. Clinical differences between the patients with CDI and those not tested and not suspected of having the infection may explain the different conclusions regarding the acid suppression effect on CDI risk.     

The Dual-Specificity Phosphatase 2 (DUSP2) Does Not Regulate Obesity-Associated Inflammation or Insulin Resistance in Mice

Alterations in the immune cell profile and the induction of inflammation within adipose tissue are a hallmark of obesity in mice and humans. Dual-specificity phosphatase 2 (DUSP2) is widely expressed within the immune system and plays a key role promoting immune and inflammatory responses dependent on mitogen-activated protein kinase (MAPK) activity. We hypothesised that the absence of DUSP2 would protect mice against obesity-associated inflammation and insulin resistance. Accordingly, male and female littermate mice that are either wild-type (wt) or homozygous for a germ-line null mutation of the dusp2 gene (dusp2^−/−) were fed either a standard chow diet (SCD) or high fat diet (HFD) for 12 weeks prior to metabolic phenotyping. Compared with mice fed the SCD, all mice consuming the HFD became obese, developed glucose intolerance and insulin resistance, and displayed increased macrophage recruitment and markers of inflammation in epididymal white adipose tissue. The absence of DUSP2, however, had no effect on the development of obesity or adipose tissue inflammation. Whole body insulin sensitivity in male mice was unaffected by an absence of DUSP2 in response to either the SCD or HFD; however, HFD-induced insulin resistance was slightly, but significantly, reduced in female dusp2^−/− mice. In conclusion, DUSP2 plays no role in regulating obesity-associated inflammation and only a minor role in controlling insulin sensitivity following HFD in female, but not male, mice. These data indicate that rather than DUSP2 being a pan regulator of MAPK dependent immune cell mediated inflammation, it appears to differentially regulate inflammatory responses that have a MAPK component.     

Deletion of RAGE Causes Hyperactivity and Increased Sensitivity to Auditory Stimuli in Mice

The receptor for advanced glycation end-products (RAGE) is a multi-ligand receptor that belongs to the immunoglobulin superfamily of cell surface receptors. In diabetes and Alzheimer''s disease, pathological progression is accelerated by activation of RAGE. However, how RAGE influences gross behavioral activity patterns in basal condition has not been addressed to date. In search for a functional role of RAGE in normal mice, a series of standard behavioral tests were performed on adult RAGE knockout (KO) mice. We observed a solid increase of home cage activity in RAGE KO. In addition, auditory startle response assessment resulted in a higher sensitivity to auditory signal and increased prepulse inhibition in KO mice. There were no significant differences between KO and wild types in behavioral tests for spatial memory and anxiety, as tested by Morris water maze, classical fear conditioning, and elevated plus maze. Our results raise a possibility that systemic therapeutic treatments to occlude RAGE activation may have adverse effects on general activity levels or sensitivity to auditory stimuli.     

Altered Glucose Homeostasis in Mice with Liver-specific Deletion of Src Homology Phosphatase 2

The Src homology 2 domain-containing protein-tyrosine phosphatase Shp2 has been implicated in a variety of growth factor signaling pathways, but its role in insulin signaling has remained unresolved. In vitro studies suggest that Shp2 is both a negative and positive regulator of insulin signaling, although its physiological function in a number of peripheral insulin-responsive tissues remains unknown. To address the metabolic role of Shp2 in the liver, we generated mice with either chronic or acute hepatic Shp2 deletion using tissue-specific Cre-LoxP and adenoviral Cre approaches, respectively. We then analyzed insulin sensitivity, glucose tolerance, and insulin signaling in liver-specific Shp2-deficient and control mice. Mice with chronic Shp2 deletion exhibited improved insulin sensitivity and increased glucose tolerance compared with controls. Acute Shp2 deletion yielded comparable results, indicating that the observed metabolic effects are directly caused by the lack of Shp2 in the liver. These findings correlated with, and were most likely caused by, direct dephosphorylation of insulin receptor substrate (IRS)1/2 in the liver, accompanied by increased PI3K/Akt signaling. In contrast, insulin-induced ERK activation was dramatically attenuated, yet there was no effect on the putative ERK site on IRS1 (Ser⁶¹²) or on S6 kinase 1 activity. These studies show that Shp2 is a negative regulator of hepatic insulin action, and its deletion enhances the activation of PI3K/Akt pathway downstream of the insulin receptor.     

Sleep Deprivation in the Dark Period Does Not Impair Memory in OF1 Mice

There is increasing evidence that sleep facilitates memory acquisition and consolidation. Moreover, the sleep-wake history preceding memory acquisition and retention as well as circadian timing may be important. We showed previously that sleep deprivation (SD) following learning in OF1 mice impaired their performance on an object recognition task. The learning task was scheduled at the end of the 12 h dark period and the test 24 h later. To investigate the influence of the prominent circadian sleep-wake distribution typical for rodents, we now scheduled the learning task at the beginning of the dark period. Wakefulness following immediately after the learning task was attained either by gentle interference (SD; n?=?20) or by spontaneous wheel running (RW; n?=?20). Two control groups were used: one had no RW throughout the experiment (n?=?23), while the other group's wheel was blocked immediately after acquisition (n?=?16), thereby preventing its use until testing. Recognition memory, defined as the difference in exploration of a novel and of familiar objects, was assessed 24 h later during the test phase. Motor activity and RW use were continuously recorded. Remarkably, performance on the object recognition task was not influenced by the protocols; the waking period following acquisition did not impair memory, independent of the method inducing wakefulness (i.e., sleep deprivation or spontaneous running). Thus, all groups explored the novel object significantly longer than the familiar ones during the test phase. Interestingly, neither the amount of rest lost during the SD interventions nor the amount of rest preceding acquisition influenced performance. However, the total amount of rest obtained by the control and SD mice subjected to acquisition at “dark offset” correlated positively (r?=?0.66) with memory at test, while no such relationship occurred in the corresponding groups tested at dark onset. Neither the amount of running nor intermediate rest correlated with performance at test in the RW group. We conclude that interfering with sleep during the dark period does not affect object recognition memory consolidation.     

Deletion of IL-18 Expression Ameliorates Spontaneous Kidney Failure in MRLlpr Mice

The role of IL-18 in the pathogenesis of systemic lupus erythematosus is still not definitively solved. In this study, we generated MRL^lpr mice, which develop a disease resembling systemic lupus erythematosus, genetically devoid of IL-18 expression. These mice in comparison to IL-18-competent MRL^lpr mice show reduced signs of renal pathogenesis, while other parameters such as mean survival time, lymphadenopathy, constitutive interferon-γ production, and frequency of CD3⁺B220⁺ abnormal T cells were without differences. We conclude that in the systemic lupus erythematosus syndrom IL-18 is involved specifically in the renal pathogenesis.     

Loss of Bace1 in Mice Does Not Alter the Severity of Caerulein Induced Pancreatitis

ContextBeta-site alpha-amyloid protein cleaving enzyme1 (BACE1) plays a key role in the pathogenesis of Alzheimer’s disease. Additional to its moderate expression in the brain, high levels of BACE1 mRNA were found in the pancreas. Murine Bace1 has been immunohistochemicaly detected at the apical pole of acinar cells within the exocrine pancreas of mice and Bace1 activity was observed in pancreatic juice. In vitro experiments revealed enteropeptidase as a putative substrate for Bace1 suggesting a role in acute pancreatitis.ObjectiveThe aim of this study was to address a protective mechanism of Bace1 in acute experimental pancreatitis in mice.MethodsAcute experimental pancreatitis was induced by intraperitoneal injection of caerulein in homozygote Bace1^-/- mice and wild type mice. Serum and tissue analyses were carried out after 4 h, 8 h and 24 h. Measurement of plasma amylase and lipase was performed to confirm pancreatitis induction. In order to assess the severity of pancreatitis H&E stained pancreatic sections were examined regarding edema, inflammation and apoptosis. Immunohistochemical detection of myeloperoxidase (MPO) positive cells was carried out to further quantify the extent of inflammation. Expression of Bace2 within the pancreas was analyzed by immunohistochemistry and RT-qPCR.ResultsWe demonstrate that total loss of Bace1 in mice leads to no alterations in the course of acute experimental caerulein-pancreatitis. Bace1^-/- mice develop a moderate pancreatitis that is comparable in histomorphological and serological features with those seen in wild type mice.DiscussionWe discuss the results in the context of the applied caerulein induced edematous pancreatitis model and possible compensatory mechanisms via Bace2 that might be responsible for the observed results.     

Recombinant Neuregulin 1 Does Not Activate Cardiomyocyte DNA Synthesis in Normal or Infarcted Adult Mice

Objectives

Neuregulin 1 signaling plays an important role in cardiac trabecular development, and in sustaining functional integrity in adult hearts. Treatment with neuregulin 1 enhances adult cardiomyocyte differentiation, survival and/or function in vitro and in vivo. It has also been suggested that recombinant neuregulin 1β1 (NRG1β1) induces cardiomyocyte proliferation in normal and injured adult hearts. Here we further explore the impact of neuregulin 1 signaling on adult cardiomyocyte cell cycle activity.

Methods and Results

Adult mice were subjected to 9 consecutive daily injections of recombinant NRG1β1 or vehicle, and cardiomyocyte DNA synthesis was quantitated via bromodeoxyuridine (BrdU) incorporation, which was delivered using mini-osmotic pumps over the entire duration of NRG1β1 treatment. NRG1β1 treatment inhibited baseline rates of cardiomyocyte DNA synthesis in normal mice (cardiomyocyte labelling index: 0.019±0.005% vs. 0.003±0.001%, saline vs. NRG1β1, P<0.05). Acute NRG1β1 treatment did result in activation of Erk1/2 and cardiac myosin regulatory light chain (down-stream mediators of neuregulin signalling), as well as activation of DNA synthesis in non-cardiomyocytes, validating the biological activity of the recombinant protein. In other studies, mice were subjected to permanent coronary artery occlusion, and cardiomyocyte DNA synthesis was monitored via tritiated thymidine incorporation which was delivered as a single injection 7 days post-infarction. Daily NRG1β1 treatment had no impact on cardiomyocyte DNA synthesis in the infarcted myocardium (cardiomyocyte labelling index: 0.039±0.011% vs. 0.027±0.021%, saline vs. NRG1β1, P>0.05).

Summary

These data indicate that NRG1β1 treatment does not increase cardiomyocyte DNA synthesis (and consequently does not increase the rate of cardiomyocyte renewal) in normal or infarcted adult mouse hearts. Thus, any improvement in cardiac structure and function observed following neuregulin treatment of injured hearts likely occurs independently of overt myocardial regeneration.     

Deletion of TAK1 in the Myeloid Lineage Results in the Spontaneous Development of Myelomonocytic Leukemia in Mice

Previous studies of the conditional ablation of TGF-β activated kinase 1 (TAK1) in mice indicate that TAK1 has an obligatory role in the survival and/or development of hematopoietic stem cells, B cells, T cells, hepatocytes, intestinal epithelial cells, keratinocytes, and various tissues, primarily because of these cells’ increased apoptotic sensitivity, and have implicated TAK1 as a critical regulator of the NF-κB and stress kinase pathways and thus a key intermediary in cellular survival. Contrary to this understanding of TAK1’s role, we report a mouse model in which TAK1 deletion in the myeloid compartment that evoked a clonal myelomonocytic cell expansion, splenomegaly, multi-organ infiltration, genomic instability, and aggressive, fatal myelomonocytic leukemia. Unlike in previous reports, simultaneous deletion of TNF receptor 1 (TNFR1) failed to rescue this severe phenotype. We found that the features of the disease in our mouse model resemble those of human chronic myelomonocytic leukemia (CMML) in its transformation to acute myeloid leukemia (AML). Consequently, we found TAK1 deletion in 13 of 30 AML patients (43%), thus providing direct genetic evidence of TAK1’s role in leukemogenesis.     

Podocyte-Specific Deletion of Murine CXADR Does Not Impair Podocyte Development,Function or Stress Response

The coxsackie- and adenovirus receptor (CXADR) is a member of the immunoglobulin protein superfamily, present in various epithelial cells including glomerular epithelial cells. Beside its known function as a virus receptor, it also constitutes an integral part of cell-junctions. Previous studies in the zebrafish pronephros postulated a potential role of CXADR for the terminal differentiation of glomerular podocytes and correct patterning of the elaborated foot process architecture. However, due to early embryonic lethality of constitutive Cxadr knockout mice, mammalian data on kidney epithelial cells have been lacking. Interestingly, Cxadr is robustly expressed during podocyte development and in adulthood in response to glomerular injury. We therefore used a conditional transgenic approach to elucidate the function of Cxadr for podocyte development and stress response. Surprisingly, we could not discern a developmental phenotype in podocyte specific Cxadr knock-out mice. In addition, despite a significant up regulation of CXADR during toxic, genetic and immunologic podocyte injury, we could not detect any impact of Cxadr on these injury models. Thus these data indicate that in contrast to lower vertebrate models, mammalian podocytes have acquired molecular programs to compensate for the loss of Cxadr.     

Adipose Overexpression of Heme Oxygenase-1 Does Not Protect against High Fat Diet-Induced Insulin Resistance in Mice

Heme oxygenase-1 (HO-1) is a stress-responsive enzyme with potent anti-oxidant and anti-inflammatory activities. Previous studies have shown that systemic induction of HO-1 by chemical inducers reduces adiposity and improves insulin sensitivity. To dissect the specific function of HO-1 in adipose tissue, we generated transgenic mice with adipose HO-1 overexpression using the adipocyte-specific aP2 promoter. The transgenic (Tg) mice exhibit similar metabolic phenotype as wild type (WT) control under chow-fed condition. High fat diet (HFD) challenge significantly increased the body weights of WT and Tg mice to a similar extent. Likewise, HFD-induced glucose intolerance and insulin resistance were not much different between WT and Tg mice. Analysis of the adipose tissue gene expression revealed that the mRNA levels of adiponectin and interleukin-10 were significantly higher in chow diet-fed Tg mice as compared to WT counterparts, whereas HFD induced downregulation of adiponectin gene expression in both Tg and WT mice to a similar level. HFD-induced proinflammatory cytokine expression in adipose tissues were comparable between WT and transgenic mice. Nevertheless, immunohistochemistry and gene expression analysis showed that the number of infiltrating macrophages with preferential expression of M2 markers was significantly higher in the adipose tissue of obese Tg mice than WT mice. Further experiment demonstrated that myeloid cells from Tg mice expressed higher level of HO-1 and exhibited greater migration response toward chemoattractant in vitro. Collectively, these data indicate that HO-1 overexpression in adipocytes does not protect against HFD-induced obesity and the development of insulin resistance in mice.     

Deletion of Protein Tyrosine Phosphatase 1B (PTP1B) Enhances Endothelial Cyclooxygenase 2 Expression and Protects Mice from Type 1 Diabetes-Induced Endothelial Dysfunction

Protein tyrosine phosphatase 1B (PTP1B) dephosphorylates receptors tyrosine kinase and acts as a molecular brake on insulin signaling pathway. Conditions of metabolic dysfunction increase PTP1B, when deletion of PTP1B protects against metabolic disorders by increasing insulin signaling. Although vascular insulin signaling contributes to the control of glucose disposal, little is known regarding the direct role of PTP1B in the control of endothelial function. We hypothesized that metabolic dysfunctions increase PTP1B expression in endothelial cells and that PTP1B deletion prevents endothelial dysfunction in situation of diminished insulin secretion. Type I diabetes (T1DM) was induced in wild-type (WT) and PTP1B-deficient mice (KO) with streptozotocin (STZ) injection. After 28 days of T1DM, KO mice exhibited a similar reduction in body weight and plasma insulin levels and a comparable increase in glycemia (WT: 384±20 vs. Ko: 432±29 mg/dL), cholesterol and triglycerides, as WT mice. T1DM increased PTP1B expression and impaired endothelial NO-dependent relaxation, in mouse aorta. PTP1B deletion did not affect baseline endothelial function, but preserved endothelium-dependent relaxation, in T1DM mice. NO synthase inhibition with L-NAME abolished endothelial relaxation in control and T1DM WT mice, whereas L-NAME and the cyclooxygenases inhibitor indomethacin were required to abolish endothelium relaxation in T1DM KO mice. PTP1B deletion increased COX-2 expression and PGI₂ levels, in mouse aorta and plasma respectively, in T1DM mice. In parallel, simulation of diabetic conditions increased PTP1B expression and knockdown of PTP1B increased COX-2 but not COX-1 expression, in primary human aortic endothelial cells. Taken together these data indicate that deletion of PTP1B protected endothelial function by compensating the reduction in NO bioavailability by increasing COX-2-mediated release of the vasodilator prostanoid PGI₂, in T1DM mice.     

Sulphatation Does Not Appear to Be a Protective Mechanism to Prevent Oxysterol Accumulation in Humans and Mice

24S- and 27-hydroxycholesterol (24OHC and 27OHC) are potent regulators of different biochemical systems in vitro and are the major circulating oxysterols. A small fraction of these oxysterols has been reported to be sulphated but there are no detailed studies. We considered the possibility that sulphatation is a protective mechanism preventing accumulation of free oxysterols. Using an accurate assay we found the sulphated fraction of 24OHC and 27OHC in circulation of adults to be less than 15% of total. In two patients with a mutation in CYP7B1 and markedly increased levels of 27OHC the sulphated fraction was 8% and 10% respectively. Infants with severe neonatal cholestasis had however markedly increased sulphate fraction of the above oxysterols. In untreated mice the degree of sulphatation of 24OHC and 27OHC in serum varied between 0 and 16%. Similar degree of sulphatation was found in two mouse models with markedly increased levels of 27OHC and 24OHC respectively. Bile duct ligated mice had higher levels of oxysterols than sham-operated controls but the sulphate fraction was not increased. We conclude that a primary increase in the levels of the oxysterols due to increased synthesis or reduced metabolism in adults and mice does not induce increased sulphatation.