Gert-Jan C.M. van den Bemd, PhD

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         Both the estrogen receptor (ER) and vitamin D receptor (VDR)  belong to the same superfamily of nuclear receptors and both receptors  are activated by interaction with their ligands. Via a complex cascade  of events already discussed in Chapter 1 of the thesis this interaction  will eventually lead to regulation of target gene transcription.  Conformational changes and phosphorylation of the receptor, as well as  interaction with dimer-partners, co-factors, basal transcription  factors, and specific sites in the DNA are major events in this cascade.
         
         1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3) and 17-oestradiol (E2),  the natural ligands for VDR and ER, respectively, have potential  therapeutic properties. However, their use as therapeutic agents is limited  because of certain side effects (Chapter 1, Sections 1.3.1 and 1.6.1 of  the thesis). The search for compounds with a more beneficial  therapeutic profile initialized the development of synthetic ligands for  VDR (1,25-(OH)2D3 analogs) and ER (SERMs). In case of VDR ligands this  means that the dissociation between calcemic effects and effects on cell  proliferation and differentiation is enlarged, in case of ER ligands  this means that the beneficial agonistic effects of E2 (e.g. maintenance  of bone mass, improved cognitive function) are mimicked, while  undesirable effects (e.g. increased risk for breast and uterine cancer)  are antagonized. Indeed, several of the newly synthesized compounds have  improved characteristics compared to the natural ligands. An  explanation for the changed biological profile is not completely clear  and obviously not similar for all synthetic ligands. It probably lies in  specific interactions with one or more of the events in the cascade  mentioned above (see also Sections 1.3.3. and 1.6.3. of the thesis).
         
         The aim of the studies presented in this thesis was to extend  the knowledge on the mechanism of action of the VDR and ER, and their  modified ligands in particular. Therefore we evaluated the differences  in effect of 1,25-(OH)2D3 and a selection of its analogs on  differentiation and growth of osteoblast-like cells and on in vitro bone  resorption (Chapter 2 of the thesis), and investigated the role of VDR  affinity (Chapter 2), conformational change of the VDR (Chapter 3), VDR  stability (Chapter 3), VDRE binding (Chapter 4), and metabolism of  1,25-(OH)2D3 analogs (Chapter 5). In Chapter 6 the effect of a selection  of SERMs on ER conformation was studied.
         
         THE VITAMIN D RECEPTOR, 1,25-DIHYDROXYVITAMIN D3, AND 1,25-DIHYDROXVITAMIN D3 ANALOGS
         The first part of this thesis focusses on ligand interaction  with the VDR. Strikingly, most investigations studying 1,25-(OH)2D3  analogs do not use cells of bone origin - a classic target tissue - but  rather nonclassic target cells like breast cancer cells and cells of the  immune system. However, in our view also the effects on bone should be  evaluated to establish an accurate biological profile of the analogs.  Therefore, the rat osteoblast-like cell line ROS 17/2.8 and the human  osteoblast-like cell line MG-63 was used as a model to evaluate the  effect of several side chain-modified 1,25-(OH)2D3 analogs on growth and  differentiation of these cells and to compare their potency with that  of 1,25-(OH)2D3. Furthermore, isolated long bones from fetal mice were  used to study the capacity of 1,25-(OH)2D3 and the analogs to release  calcium from bone.
         
         
         EFFECTS ON CELL GROWTH AND DIFFERENTIATION AND ON IN VITRO BONE RESORPTION
         All 1,25-(OH)2D3 analogs examined in this thesis exhibited  agonistic activity in all responses tested: like 1,25-(OH)2D3 the  analogs stimulated the synthesis of the bone matrix proteins osteocalcin  and type I procollagen. Furthermore, the analogs mimicked the natural  ligand in their effect on osteoblast-like cell growth and in their  stimulatory effect on in vitro bone resorption (Chapter 2 of the  thesis). Some analogs (OCT, MC903, CB966) exhibited an in vitro  biological potency comparable to that of 1,25-(OH)2D3, whereas others  (KH1049, KH1060, EB1089) were much more potent than 1,25-(OH)2D3. This  observation is supported by almost every in vivo and in vitro study  published so far: 1,25-(OH)2D3 analogs are super, full, or partial  agonists of 1,25-(OH)2D3.
         
         We found that the potency of the 1,25-(OH)2D3 analogs was  related to the biological response. In some responses their potency  (compared to 1,25-(OH)2D3) was only moderately increased, while in other  responses the increase in potency was much more striking. In Table 2.3  of the thesis this differential effect on gene regulation is expressed  as the ED50 ratio for stimulation of in vitro bone resorption and  stimulation of extracellular bone matrix protein synthesis. It clearly  demonstrates that for EB1089 and KH1060 the ED50 ratio is increased  compared to the ED50 ratio for 1,25-(OH)2D3. The finding that for some  analogs the ED50 ratio between bone formation parameters (stimulation of  osteocalcin and type I collagen synthesis) and bone resorption  parameters (stimulation of calcium release) is increased compared to the  ED50 ratio for 1,25-(OH)2D3 might support a therapeutic potential of  these analogs in the treatment of metabolic bone diseases, i.e.  osteoporosis. A relationship between increased bone turnover and the  risk for metastasis of cancer to bone has been reported. The in vivo  potency of these analogs to induce bone resorption must therefore be  examined with great care.
         
         The studies presented in Chapter 2 of the thesis show cell  type-specific differences in sensitivity for 1,25-(OH)2D3 and some of  the analogs. For instance, in MG-63 cells maximal stimulation of  osteocalcin production by EB1089, KH1049, and KH1060 was almost two-fold  increased compared to 1,25-(OH)2D3 and the analogs with moderate  activity (OCT, MC903, CB966), while no difference in maximal stimulation  was observed in ROS 17/2.8 cells. However, in general, MG-63 cells were  considerably less sensitive than ROS 17/2.8 cells. For instance, in  MG-63 cells the ED50 for osteocalcin synthesis induced by 1,25-(OH)2D3,  OCT, CB966, or EB1089 was reached at 100-600 times higher concentrations  than in ROS 17/2.8 cells. Cell type-specific differences in VDR content  and/or function might underlie this phenomenon. We found that MG-63  cells have a much lower VDR content compared to ROS 17/2.8 cells and do  not show homologous VDR up-regulation (data  not shown). In addition, cell type-specific differences in presence  and/or distribution of cofactors might play a role. We also observed  that the ED50’s for both KH1049 and KH1060 (both 20-epi analogs) were  comparable between the two cell lines, indicating that for these analogs  cell type-specific differences might be of less importance. Another  possibility is that KH1049 and KH1060 are metabolized more efficiently  by ROS 17/2.8 cells, (or less efficiently by MG-63 cells) in relation to  1,25-(OH)2D3 and the other analogs. This could lead to relatively high  concentrations of KH analogs in MG-63 cells or relatively low  concentrations of these compounds in ROS 17/2.8 cells.
         
         With most cell types 1,25-(OH)2D3 has an inhibitory effect on  growth; a feature that gave rise to the thought to use 1,25-(OH)2D3 or  its analogs for the treatment of hyperproliferative diseases. Likewise,  with the human osteosarcoma cell line MG-63 and the human breast cancer  cell lines MCF-7 and ZR75-1 our laboratory observed dose-dependent  inhibition of proliferation by 1,25-(OH)2D3 and its analogs (Chapter 2  of the thesis). However, the rat osteblast-like ROS 17/2.8 cells were  stimulated in their growth. Interestingly, the analogs exhibited a  similar order of potency in their growth inhibitory activity (in MG-63  cells) and in their growth stimulatory activity (in ROS 17/2.8 cells).
         
         
         VDR CONFORMATION AND VDR STABILITY
         One of the key events in the cascade leading to regulation of  gene transcription is the ligand-induced conformational change of the  VDR. To study the effects of 1,25-(OH)2D3 and KH1060 on VDR conformation  partial proteolytic digestion  analysis was used. The method is based on the idea that accessibility  of potential protease cleavage sites within a receptor molecule alters  as a result of ligand-induced changes in receptor conformation.  Consequently, changes in receptor conformation will lead to changes in  the protease digestion pattern of the receptors. We found that in the  absence of ligand in vitro synthesized VDR is rapidly degraded by  proteases. In contrast, 1,25-(OH)2D3 and the analogs changed the VDR  conformation, resulting in enhanced protease resistance of distinct VDR  fragments. In general 1,25-(OH)2D3 analogs can be regarded as agonists,  although the potency by which they mimic the natural ligand differs  between tissues, cells, and responses studied. This probably explains  why we could not establish qualitative differences in the VDR protease  digestion profiles of 1,25-(OH)2D3 and the analogs: the sizes of the  protease resistant VDR fragments were not distinct, only the intensity  of the preserved fragments was different.
         
         Interestingly, we and others found that treatment of unliganded  in vitro synthesized VDR with low trypsin concentrations resulted in  preservation of VDR fragments with a similar size as the fragments  observed with liganded VDR (Chapter 3 of the thesis). When protease  digestion was intensified (by increasing the trypsin concentration or  the trypsin incubation time) these fragments disappeared, whereas the  fragments of liganded VDR were still protected. 
         
         We observed that 1,25-(OH)2D3 analogs and metabolites with  strong biological activity are also strong inducers of a VDR  conformation with increased protease resistance and vice versa (Chapters  2, 3, and 4 of the thesis). However, one should be cautious in  overestimating the quantitative differences obtained by limited protease  digestion analysis. For instance, the strong biological effect of  EB1089 (an analog with normal configuration at C-20) is not reflected by  a strong increase in the intensity of protease resistant VDR fragments  (data not shown). One can not exclude the possibility that some potent  1,25-(OH)2D3 analogs might induce a conformational change of the VDR  that does not lead to increased protease resistance but even to  increased protease sensitivity.
         
         Crystal structure analysis revealed that liganded nuclear  receptors adopt a more compact structure, obviously less sensitive to  protease activity. In cells this property could lead to increased VDR  stability. Indeed, our studies presented in Chapters 3 and 5 of the  thesis show that 1,25-(OH)2D3, analogs and specific metabolites of the  analog KH1060 prolong VDR half-life in ROS 17/2.8 cells.
         
         DNA BINDING
         The ligand-induced conformational change of the VDR probably  facilitates dimerization with RXR and enhances binding to specific  regulatory elements (VDREs) in the vicinity of 1,25-(OH)2D3 target  genes. In Chapter 4 of the thesis the potency of KH1060 was compared  with the potency of 1,25-(OH)2D3 to induce binding of the VDR to VDREs.  Both in vitro synthesized VDR/RXR as well as nuclear extracts of ROS  17/2.8 cells were used to investigate VDR binding to VDREs from three  different target genes: rat osteocalcin, human osteocalcin and mouse  osteopontin. Both 1,25-(OH)2D3 and KH1060 induced VDR binding to these  VDREs in a ligand dose-dependent manner, independent of the source of  VDRs (in vitro synthesized or nuclear extracts). All three VDREs are of  the DR3 type: two hexameric half-sites separated by three nucleotides,  and despite of minor differences in nucleotide sequence (see Table 4.1  of the thesis) large differences in VDR-VDRE affinity were observed. The  VDR bound with lowest affinity to the rat osteocalcin VDRE, and with  highest affinity to the mouse osteopontin VDRE. We further elaborated on  this interesting finding and produced hybrid VDREs in which the rat  osteocalcin VDRE half-sites were replaced by one or both mouse  osteopontin VDRE half-sites. Gel mobility shift assays performed with  these VDREs clearly showed that the proximal half-site of the mouse  osteopontin VDRE (i.e. the OC/OP VDRE) was mainly responsible for the  increased affinity. Transfection studies in ROS 17/2.8 cells revealed  that 1,25-(OH)2D3-induced transcription of the OC/OP VDRE-driven  reporter gene was more effective than via the rat osteocalcin  VDRE-driven reporter. Further substitution studies revealed that in  particular the third and/or fourth nucleotides (both thymidine)  determined the difference in VDR-VDRE affinity.
         
         We found that KH1060 was a stronger inducer of VDR/RXR binding  to the mouse osteopontin VDRE than 1,25-(OH)2D3. However, surprisingly,  when we studied binding of in vitro synthesized VDR/RXR to the human and  rat osteocalcin VDRE KH1060 exhibited no increased potency compared to  1,25-(OH)2D3. In contrast, when VDR protein was extracted from  ligand-treated cells KH1060 was a stronger inducer of VDR/RXR-VDRE  binding than 1,25-(OH)2D3 with all VDRE types tested. So, this indicates  that the KH1060-induced conformational change of the VDR does not  necessarily result in enhanced VDRE binding, but in addition depends on  the VDRE nucleotide sequence and furthermore underlines the importance  of a cellular context (e.g. presence of cofactors) to obtain optimal  VDR-VDRE binding. Therefore, it seems justified to speculate that KH1060  changes the VDR conformation in such a way that, compared to the  1,25-(OH)2D3-induced VDR conformation, cofactor binding is facilitated  or that other KH1060-specific cofactors are involved and that this will  lead to increased VDR-VDRE binding.
         
         ANALOG METABOLITES
         Formation of biologically active  metabolites might be a cause for concern. Obviously, from a therapeutic  point of view rapidly acting analogs with inactive metabolites are much  better to control than analogs that are metabolized into one or more  active metabolites. For each of these metabolites the mechanism of  action and possible side effects should be established before safe  clinical use can be guaranteed. Chapter 5 of the thesis focusses on the  potential contribution of metabolites of KH1060 to the biological  potency of the parent compound. Earlier was shown that in vitro KH1060  is metabolized into at least 22 different compounds. The metabolites  24a-OH-KH1060, 26-OH-KH1060, and 26a-OH-KH1060 were potent inducers of  osteopontin mRNA expression, rat osteocalcin VDRE-driven reporter gene  transcription, and active stimulators of osteocalcin synthesis (Chapter 5  of the thesis). The same metabolites were also active inducers of VDR  binding to rat osteocalcin and rat cytochrome P450 (CYP24) VDREs  (Chapter 5). Next, we investigated whether the biological potency of  these metabolites was correlated with their potency to induce a  conformational change of the VDR with enhanced protease resistance.  Indeed, partial protease digestion analysis showed that the biological  active metabolites changed the VDR conformation resulting in enhanced  protease resistance, whereas a metabolite with weak biological activity  was inactive in the protease digestion analysis. A corresponding pattern  was observed studying the effect of the metabolites on VDR stability in  ROS 17/2.8 cells.
         
         THE ESTROGEN RECEPTOR, 17b-ESTRADIOL, AND SELECTIVE ESTROGEN RECEPTOR MODULATORS
         The second part of the thesis focusses on ligand interaction  with the ER. We studied the effect of E2 and several synthetic ER  ligands on the conformation of both ER subtypes: ERa and ER. One of the  tested ER ligands, tamoxifen, is a SERM with tissue-dependent partial  agonistic/antagonistic activities: it mimics E2 with anabolic effects on  bone, protective effects on the cardiovascular system, and beneficial  effects on cognitive function, whereas it counteracts the stimulatory  effect of E2 on breast tumors (See Section B.5.2. of the thesis). An  explanation for the tissue-dependent partial agonistic/antagonistic  activation of SERMs is still unknown.
         
         Using partial protease digestion analysis we showed that  tamoxifen changed the conformation of both ERa and ERb differently from  that induced by E2. Also other partial agonists/antagonists  (4-hydroxytamoxifen, idoxifen, and the raloxifene derivative LY  117,018-HCl) had a similar effect as tamoxifen. Like other investigators  we could not discriminate between different SERMs based on the protease  digestion profiles. Other techniques, like phage enzyme-linked  immunoassays have shown to be more suitable for this purpose.  Furthermore, it is difficult to translate the intensity of the preserved  fragments obtained with the partial protease digestion analysis into a  functional receptor affinity as was done for VDR analogs. Therefore,  protease resistance (as a tool to study ligand-induced conformational  changes of a receptor) can only be used to calculate a functional  receptor affinity when the investigated ligands exert agonistic  activities. However, for nuclear hormone receptors like the progesterone  receptor and ER (partial) agonistic/antagonistic ligands are known.  Interaction with these ligands lead to a conformational change of the  receptor that is clearly distinguishable by partial digestion analysis  from the conformational change induced by ligands with pure agonistic  action.         

Articles

Both the estrogen receptor (ER) and vitamin D receptor (VDR) belong to the same superfamily of nuclear receptors and both receptors are activated by interaction with their ligands.

Author | Gert-Jan C.M. van den Bemd, PhD

Articles

Studies on the Mechanism of Action of Synthetic Ligands of the Vitamin D and Estrogen receptor