Todd McMullen

New aspects of the interaction of cholesterol with dipalmitoylphosphatidylcholine bilayers as revealed by high-sensitivity differential scanning calorimetry

We have investigated the effects of cholesterol on the thermotropic phase behavior of annealed and unannealed aqueous dispersions of dipalmitoylphosphatidylcholine (DPPC) using high-sensitivity differential scanning calorimetry (DSC), concentrating particularly on the cholesterol concentration range from 0 to 20 mol%. We find that the incorporation of cholesterol into low-temperature annealed DPPC bilayers decreases the enthalpy of the subtransition without affecting the transition temperature, such that the subtransition is abolished by 20 mol% cholesterol. Similarly, the incorporation of cholesterol progressively decreases the temperature and enthalpy of the pretransition and abolishes it entirely at cholesterol concentrations above 5 mol%. The incorporation of increasing quantities of cholesterol also alters the main or chain-melting phase transition. At cholesterol concentrations of 2 to 20 mol% cholesterol, the DSC endotherm arising from the main transition consists of superimposed sharp and broad components, the former due to the melting of cholesterol-poor and the latter to the melting of the cholesterol-rich DPPC domains. The temperature and cooperativity of the sharp component decreases slightly with increasing cholesterol concentration whereas the enthalpy decreases markedly, becoming zero at 20-25 mol% cholesterol. In contrast, the temperature and enthalpy of the broad component increases, and the cooperativity decreases markedly over this same range of cholesterol concentrations. An apparent increase in cooperativity of the overall DPPC endotherm at 7 mol% cholesterol is shown to arise because of a convergence in the transition temperatures of the sharp and broad components of the DSC endotherms. Some of our experimental findings, particularly the absence of any evidence for the existence of a triple point near 7.5 mol% cholesterol, do not accord with a recently proposed DPPC/cholesterol phase diagram derived from DSC and 2H-NMR data (see Vist, M.R. and Davis, J.H. (1990) Biochemistry 29, 451-464). In addition, we examined the effect of cholesterol on phosphatidylcholines (PCs) of different chain lengths and confirm that a eutectic point does not exist for any of these PC/cholesterol mixtures. We then propose a new, more complete DPPC/cholesterol phase diagram based on our high-sensitivity DSC data as well as some recent spectroscopic data on PC/cholesterol mixtures and explore some of its biological implications.

Physical studies of cholesterol-phospholipid interactions

Our understanding of the role of cholesterol in biological membranes requires a detailed knowledge of the forces which mediate cholesterol-lipid interactions and of the lateral organization of cholesterol in natural and model membranes. The recent literature amply illustrates the strength of a multidisciplinary approach in determining the effects of cholesterol on the host bilayer lipids. To confidently apply our current knowledge of cholesterol-lipid interactions and cholesterol organization to natural membranes, however, a systematic analysis of the effects of cholesterol on a much broader range of host lipid bilayers will be required.

The effect of variations in phospholipid and sterol structure on the nature of lipid–sterol interactions in lipid bilayer model membranes

This review deals with the effect of variations in phospholipid and sterol structure on the nature and magnitude of lipid-sterol interactions in lipid bilayer model membranes. The first portion of the review covers the effect of Chol itself on the thermotropic phase behavior and organization of a variety of different glycero- and sphingolipid membrane lipid classes, varying in the structure and charge of their polar headgroups and in the length and structure of their fatty acyl chains. The second part of this review deals with the effect of variations in sterol structure on the thermotropic phase behavior and organization primarily of the well studied DPPC model membrane system. In the third section, we focus on some of the contributions of sterol functional group chemistry, molecular conformation and dynamics, to sterol-lipid interactions. Using those studies, we re-examine the results of recently published experimental and computer-modeling studies to provide a new more dynamic molecular interpretation of sterol-lipid interactions. We suggest that the established view of the rigid sterol ring system and extended alkyl side-chain obtained from physical studies of cholesterol-phospholipid mixtures may not apply in lipid mixtures differing in their sterol chemical structure.

Calorimetric and spectroscopic studies of the effects of cholesterol on the thermotropic phase behavior and organization of a homologous series of linear saturated phosphatidylglycerol bilayer membranes.

We have examined the effects of cholesterol (Chol) on the thermotropic phase behavior and organization of aqueous dispersions of a homologous series of linear disaturated phosphatidylglycerols (PGs) by high-sensitivity differential scanning calorimetry and Fourier transform infrared and 31P NMR spectroscopy. We find that the incorporation of increasing quantities of Chol alters the temperature and progressively reduces the enthalpy and cooperativity of the gel-to-liquid-crystalline phase transition of the host PG bilayer. With dimyristoyl-PG:Chol mixtures, cooperative chain-melting phase transitions are completely or almost completely abolished at Chol concentrations near 50 mol%, whereas with the dipalmitoyl- and distearoyl-PG:Chol mixtures, cooperative hydrocarbon chain-melting phase transitions are still discernable at Chol concentrations near 50 mol%. We are also unable to detect the presence of significant populations of separate domains of the anhydrous or monohydrate forms of Chol in our binary mixtures, in contrast to previous reports. We ascribe the previously reported large scale formation of Chol crystallites to the fractional crystallization of the Chol and phospholipid phases during the removal of organic solvent from the binary mixture before the hydration of the sample. We further show that the direction and magnitude of the change in the phase transition temperature induced by Chol addition is dependent on the hydrocarbon chain length of the PG studied. This finding agrees with our previous results with phosphatidylcholine bilayers, where we found that Chol increases or decreases the phase transition temperature in a hydrophobic mismatch-dependent manner (Biochemistry 1993, 32:516-522), but is in contrast to our previous results for phosphatidylethanolamine (Biochim. Biophys. Acta 1999, 1416:119-234) and phosphatidylserine (Biophys. J. 2000, 79:2056-2065) bilayers, where no such hydrophobic mismatch-dependent effects were observed. We also show that the addition of Chol facilitates the formation of the lamellar crystalline phase in PG bilayers, as it does in phosphatidylethanolamine and phosphatidylserine bilayers, whereas the formation of such phases in phosphatidylcholine bilayers is inhibited by the presence of Chol. Moreover, the formation of the lamellar crystalline phase in PG bilayers at lower temperatures excludes Chol, resulting in an apparent Chol immiscibility in gel-state PG bilayers. We suggest that the magnitude of the effect of Chol on the thermotropic phase behavior of the host phospholipid bilayer, and its miscibility in phospholipids dispersions generally, depend on the strength of the attractive interactions between the polar headgroups and the hydrocarbon chains of the phospholipid molecule, and not on the charge of the polar headgroups per se.

Comparative differential scanning calorimetric and FTIR and 31P-NMR spectroscopic studies of the effects of cholesterol and androstenol on the thermotropic phase behavior and organization of phosphatidylcholine bilayers

We have investigated the comparative effects of the incorporation of increasing quantities of androstenol and cholesterol on the thermotropic phase behavior of aqueous dispersions of members of a homologous series of linear saturated diacyl PCs1 using high sensitivity DSC. We have also employed FTIR and 31P-NMR spectroscopy to study the comparative effects of androstenol and cholesterol incorporation on the organization of the host PC bilayer in both the gel and liquid-crystalline states. The effects of androstenol and cholesterol incorporation on the thermotropic phase behavior of shorter chain PCs like 14:0 PC are generally similar but not identical. The incorporation of either sterol progressively decreases the temperature and enthalpy, but not the cooperativity, of the pretransition and completely abolishes it at sterol concentrations above 5 mol%. Moreover, at sterol concentrations of 1 to 20-25 mol%, both androstenol and cholesterol incorporation produce DSC endotherms consisting of superimposed sharp and broad components, the former due to the hydrocarbon chain melting of sterol-poor and the latter to the melting of sterol-rich 14:0 PC domains. The temperature and cooperativity of the sharp component are reduced slightly with increasing concentration of androstenol or cholesterol, and the enthalpy of the sharp component decreases progressively and becomes zero at 20-25 mol% sterol. As well, at cholesterol or androstenol concentrations above 20-25 mol%, the enthalpy of the broad component also decreases linearly with increasing sterol incorporation and becomes zero at sterol levels of about 50 mol%. However, whereas cholesterol incorporation progressively increases the temperature of the broad component of the DSC endotherm, androstenol incorporation decreases the temperature of this component. In contrast, the effects of androstenol and cholesterol incorporation on the thermotropic phase behavior of the intermediate and longer chain PCs studied here are considerably different. Although the incorporation of cholesterol increases the main phase transition temperature of 16:0 PC slightly and decreases the phase transition of 18:0 PC and 21:0 PC, androstenol incorporation decreases the main phase transition temperatures of all three PCs rather markedly. Moreover, androstenol is less effective in reducing the enthalpy and cooperativity of the broad component of the DSC endotherm of 16:0 PC and especially 18:0 PC bilayers in comparison to cholesterol. Androstenol incorporation (> 5 mol%) also results in the appearance of a second, low temperature endotherm in the DSC traces of the intermediate and longer chain PC dispersions that is not observed in similar cholesterol/PC dispersions. FTIR and 31P-NMR results suggest that this endotherm arises from a temperature-induced dissolution of androstenol in the gel phase PC bilayers. This second endotherm occurs at lower androstenol concentrations and increases in area at a given androstenol level as the chain length of the host PC bilayer increases. We ascribe the increasing immiscibility of androstenol in both the gel and liquid-crystalline states of PC bilayers of increasing thickness to an increasing degree of hydrophobic mismatch between the androstenol molecule and the host phospholipid bilayer.

Differential scanning calorimetric and Fourier transform infrared spectroscopic studies of the effects of cholesterol on the thermotropic phase behavior and organization of a homologous series of linear saturated phosphatidylserine bilayer membranes.

We have examined the effects of cholesterol on the thermotropic phase behavior and organization of aqueous dispersions of a homologous series of linear disaturated phosphatidylserines by high-sensitivity differential scanning calorimetry and Fourier transform infrared spectroscopy. We find that the incorporation of increasing quantities of cholesterol progressively reduces the temperature, enthalpy, and cooperativity of the gel-to-liquid-crystalline phase transition of the host phosphatidylserine bilayer, such that a cooperative chain-melting phase transition is completely or almost completely abolished at 50 mol % cholesterol, in contrast to the results of previous studies. We are also unable to detect the presence of a separate anhydrous cholesterol or cholesterol monohydrate phase in our binary mixtures, again in contrast to previous reports. We further show that the magnitude of the reduction in the phase transition temperature induced by cholesterol addition is independent of the hydrocarbon chain length of the phosphatidylserine studied. This result contrasts with our previous results with phosphatidylcholine bilayers, where we found that cholesterol increases or decreases the phase transition temperature in a chain length-dependent manner (1993. Biochemistry, 32:516-522), but is in agreement with our previous results for phosphatidylethanolamine bilayers, where no hydrocarbon chain length-dependent effects were observed (1999. Biochim. Biophys. Acta, 1416:119-234). However, the reduction in the phase transition temperature by cholesterol is of greater magnitude in phosphatidylethanolamine as compared to phosphatidylserine bilayers. We also show that the addition of cholesterol facilitates the formation of the lamellar crystalline phase in phosphatidylserine bilayers, as it does in phosphatidylethanolamine bilayers, whereas the formation of such phases in phosphatidylcholine bilayers is inhibited by the presence of cholesterol. We ascribe the limited miscibility of cholesterol in phosphatidylserine bilayers reported previously to a fractional crystallization of the cholesterol and phospholipid phases during the removal of organic solvent from the binary mixture before the hydration of the sample. In general, the results of our studies to date indicate that the magnitude of the effect of cholesterol on the thermotropic phase behavior of the host phospholipid bilayer, and its miscibility in phospholipid dispersions generally, depend on the strength of the attractive interactions between the polar headgroups and the hydrocarbon chains of the phospholipid molecule, and not on the charge of the polar headgroups per se.

The effect of side-chain analogues of cholesterol on the thermotropic phase behavior of 1-stearoyl-2-oleoylphosphatidylcholine bilayers: a differential scanning calorimetric study

In this study we have examined the effects of analogues of cholesterol differing with respect to alkyl side-chain length and structure on the thermotropic phase behavior of bilayers formed from 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), an important subclass of naturally occurring phosphatidylcholines (PCs). The synthetic sterols we studied contained either a terminally unbranched (n-series) or a single methyl-branched (iso-series) side chain of 3 to 10 carbon atoms. The phase transition behavior was examined by high-sensitivity differential scanning calorimetry (DSC). The main phase transition endotherm of SOPC/sterol bilayers consists of superimposed sharp and broad components, which represent the hydrocarbon chain melting of sterol-poor and sterol-rich phospholipid domains, respectively. The transition temperature and the cooperativity of the sharp component are moderately reduced upon sterol incorporation and the enthalpy decreases to zero when sterol levels of 20-30 mol% are reached. The enthalpy of the broad component transition initially increases to a maximum around 25 or 25-30 mol% sterol and thereafter decreases with further increases in sterol concentration. However, the broad transition of SOPC bilayers containing both short (C-22, i-C5 and n-C3) and long (i-C9 and i-C10) side-chain sterols still persists at levels of 50 mol% sterol. Thus the effective stoichiometry of SOPC-sterol interactions varies with changes in sterol alkyl side-chain length. The incorporation of short linear or branched side-chain sterols (C-22, n-C3, n-C4, i-C5) causes the broad component transition temperature and cooperativity to decrease dramatically, whereas the incorporation of medium- and long-chain sterols in both the n- and iso-series has less effect on the transition temperature and cooperativity of the broad component. Overall, no significant differences were found between the n- and iso-series sterols for a given side-chain length. A comparison of the phase behavior of dipalmitoylphosphatidylcholine (DPPC)/sterol (McMullen et al. (1995) Biophys. J. 69, 169-176) and SOPC/sterol mixtures indicates that the primary factor responsible for changes in the thermotropic phase behavior of these systems is the extent of the hydrophobic mismatch between the sterol and the host lipid bilayer. However, sterol miscibility in PC bilayers, and thus the stoichiometry of lipid-sterol interactions, also appears to depend on the degree of unsaturation of the host lipid bilayer.

Differential scanning calorimetric study of the effect of sterol side chain length and structure on dipalmitoylphosphatidylcholine thermotropic phase behavior

We have investigated the thermotropic phase behavior of dipalmitoylphosphatidylcholine (DPPC) bilayers containing a series of cholesterol analogues varying in the length and structure of their alkyl side chains. We find that upon the incorporation of up to approximately 25 mol % of any of the side chain analogues, the DPPC main transition endotherm consists of superimposed sharp and broad components representing the hydrocarbon chain melting of sterol-poor and sterol-rich phospholipid domains, respectively. Moreover, the behavior of these components is dependent on sterol side chain length. Specifically, for all sterol/DPPC mixtures, the sharp component enthalpy decreases linearly to zero by 25 mol % sterol while the cooperativity is only moderately reduced from that observed in the pure phospholipid. In addition, the sharp component transition temperature decreases for all sterol/DPPC mixtures; however, the magnitude of the decrease is dependent on the sterol side chain length. With respect to the broad component, the enthalpy initially increases to a maximum around 25 mol % sterol, thereafter decreasing toward zero by 50 mol % sterol with the exception of the sterols with very short alkyl side chains. Both the transition temperature and cooperativity of the broad component clearly exhibit alkyl chain length-dependent effects, with both the transition temperature and cooperativity decreasing more dramatically for sterols with progressively shorter side chains. We ascribe the chain length-dependent effects on transition temperature and cooperativity to the hydrophobic mismatch between the sterol and the host DPPC bilayer (see McMullen, T. P. W., Lewis, R. N. A. H., and McElhaney, R. N. (1993) Biochemistry 32:516-522). Moreover, the effective stoichiometry of sterol/DPPC interactions is altered by a significantly large degree of hydrophobic mismatch between the sterol and the DPPC bilayer. Thus the short chain sterols appear to exhibit considerable immiscibility in gel state DPPC bilayers, effectively limiting their interaction with adjacent phospholipid molecules.

Inhibition of autotaxin delays breast tumor growth and lung metastasis in mice

Autotaxin is a secreted enzyme that produces most extracellular lysophosphatidate, which stimulates 6 G‐protein‐coupled receptors. Lysophosphatidate promotes cancer cell survival, growth, migration, invasion, metastasis, and resistance to chemotherapy and radiotherapy. The present work investigated whether inhibiting autotaxin could decrease breast tumor growth and metastasis. We used a new autotaxin inhibitor (ONO‐8430506; IC90=100 nM), which decreased plasma autotaxin activity by >60% and concentrations of unsaturated lysophosphatidates by >75% for 24 h compared with vehicle‐treated mice. The effects of ONO‐8430506 on tumor growth were determined in a syngeneic orthotopic mouse model of breast cancer following injection of 20,000 BALB/c mouse 4T1 or 4T1‐12B cancer cells. We show for the first time that inhibiting autotaxin decreases initial tumor growth and subsequent lung metastatic nodules both by 60% compared with vehicle‐treated mice. Significantly, 4T1 cells express negligible autotaxin compared with the mammary fat pad. Autotaxin activity in the fat pad of nontreated mice was increased 2‐fold by tumor growth. Our results emphasize the importance of tumor interaction with its environment and the role of autotaxin in promoting breast cancer growth and metastasis. We also established that autotaxin inhibition could provide a novel therapeutic approach to blocking the adverse effects of lysophosphatidate in cancer.—Benesch, M. G. K., Tang, X., Maeda, T., Ohhata, A., Zhao, Y. Y., Kok, B. P. C., Dewald, J., Hitt, M., Curtis, J. M., McMullen, T. P. W., Brindley, D. N. Inhibition of autotaxin delays breast tumor growth and lung metastasis in mice. FASEB J. 28, 2655–2666 (2014). www.fasebj.org

Autotaxin in the crosshairs: Taking aim at cancer and other inflammatory conditions

Autotaxin is a secreted enzyme that produces most of the extracellular lysophosphatidate from lysophosphatidylcholine, the most abundant phospholipid in blood plasma. Lysophosphatidate mediates many physiological and pathological processes by signaling through at least six G‐protein coupled receptors to promote cell survival, proliferation and migration. The autotaxin/lysophosphatidate signaling axis is involved in wound healing and tissue remodeling, and it drives many chronic inflammatory conditions from fibrosis to colitis, asthma and cancer. In cancer, lysophosphatidate signaling promotes resistance to chemotherapy and radiotherapy, and increases both angiogenesis and metastasis. Research into autotaxin inhibitors is accelerating, both as primary and adjuvant therapy. Historically, autotaxin inhibitors had poor bioavailability profiles and thus had limited efficacy in vivo. This situation is now changing, especially since the recent crystal structure of autotaxin is now enabling rational inhibitor design. In this review, we will summarize current knowledge on autotaxin‐mediated disease processes including cancer, and discuss recent advancements in the development of autotaxin‐targeting strategies. We will also provide new insights into autotaxin as an inflammatory mediator in the tumor microenvironment that promotes cancer progression and therapy resistance.