Alexander Kouzmenko

Dioxin receptor is a ligand-dependent E3 ubiquitin ligase

Fat-soluble ligands, including sex steroid hormones and environmental toxins, activate ligand-dependent DNA-sequence-specific transcriptional factors that transduce signals through target-gene-selective transcriptional regulation. However, the mechanisms of cellular perception of fat-soluble ligand signals through other target-selective systems remain unclear. The ubiquitin–proteasome system regulates selective protein degradation, in which the E3 ubiquitin ligases determine target specificity. Here we characterize a fat-soluble ligand-dependent ubiquitin ligase complex in human cell lines, in which dioxin receptor (AhR) is integrated as a component of a novel cullin 4B ubiquitin ligase complex, CUL4BAhR. Complex assembly and ubiquitin ligase activity of CUL4BAhR in vitro and in vivo are dependent on the AhR ligand. In the CUL4BAhR complex, ligand-activated AhR acts as a substrate-specific adaptor component that targets sex steroid receptors for degradation. Thus, our findings uncover a function for AhR as an atypical component of the ubiquitin ligase complex and demonstrate a non-genomic signalling pathway in which fat-soluble ligands regulate target-protein-selective degradation through a ubiquitin ligase complex.

Wnt and PPARγ signaling in osteoblastogenesis and adipogenesis

Osteoblasts and adipocytes differentiate from a common pluripotent precursor, the mesenchymal stem cell (MSC). Studies have identified numerous transcription factors, and multiple extracellular and intracellular signaling pathways that regulate the closely linked processes of adipogenesis and osteoblastogenesis. Interestingly, inducers of differentiation along one lineage often inhibit differentiation along the other; for example, the transcription factor peroxisome proliferator-activated receptor γ (PPARγ) is a prime inducer of adipogenesis that inhibits osteoblastogenesis. The latest research has shown that inducers of osteoblastogenesis (such as bone morphogenetic protein 2 and Wnt ligands) use different mechanisms to suppress the transactivation function of PPARγ during osteoblastogenesis from MSCs. Signaling via the canonical Wnt–β-catenin pathway inhibits PPARγ mRNA expression, whereas signaling via the noncanonical Wnt pathway results in activation of a histone methyltransferase SETDB1 that represses PPARγ transactivation through histone H3K9 methylation of target genes. This article summarizes Wnt and PPARγ signaling in MSCs and the crosstalk between these pathways, and speculates on future clinical application of this knowledge as the basis of novel approaches for regeneration therapy.

The Androgen Receptor in Health and Disease

Androgens play pivotal roles in the regulation of male development and physiological processes, particularly in the male reproductive system. Most biological effects of androgens are mediated by the action of nuclear androgen receptor (AR). AR acts as a master regulator of downstream androgen-dependent signaling pathway networks. This ligand-dependent transcriptional factor modulates gene expression through the recruitment of various coregulator complexes, the induction of chromatin reorganization, and epigenetic histone modifications at target genomic loci. Dysregulation of androgen/AR signaling perturbs normal reproductive development and accounts for a wide range of pathological conditions such as androgen-insensitive syndrome, prostate cancer, and spinal bulbar muscular atrophy. In this review we summarize recent advances in understanding of the epigenetic mechanisms of AR action as well as newly recognized aspects of AR-mediated androgen signaling in both men and women. In addition, we offer a perspective on the use of animal genetic model systems aimed at eventually developing novel therapeutic AR ligands.

Molecular switching of osteoblastogenesis versus adipogenesis: implications for targeted therapies.

Osteoblasts and adipocytes differentiate from a common precursor, the pluripotent mesenchymal stem cell (MSC) found in bone marrow (BMSC) and adipose tissue (AD-MSC). Numerous transcription factors and multiple extracellular and intracellular signals regulating adipogenesis and osteoblastogenesis have been identified and analyzed. Significantly, inducers of differentiation towards one lineage may inhibit cell differentiation into an alternative lineage. For example, the canonical Wnt/β-catenin pathway induces osteoblastogenesis and inhibits adipogenesis, whereas the peroxisome proliferator activated receptor-γ (PPAR-γ) is a prime inducer of adipogenesis and, as shown in recent studies, inhibits osteoblastogenesis. We have identified two signaling pathways that switch the cell fate decision from adipocytes to osteoblasts by suppressing the transactivation function of PPAR-γ. In the first pathway, the TNF-α- or IL-1-induced TAK1/TAB1/NIK signaling cascade attenuates PPAR-γ-mediated adipogenesis by inhibiting the binding of PPAR-γ to the DNA response element. The second is the noncanonical Wnt pathway through the CaMKII-TAK1/TAB2-NLK (nemo-like kinase) signaling cascade. Specifically, Wnt-5a-induced phosphorylation of NLK triggers formation of a complex with the histone methyltransferase SETDB1 (SET domain, bifurcated 1) that represses PPAR-γ transactivation through histone H3-K9 methylation at the target genes. Thus, two signaling cascades promote osteoblastic differentiation from MSC through two distinct modes of PPAR-γ transrepression.

Estrogens Maintain Bone Mass by Regulating Expression of Genes Controlling Function and Life Span in Mature Osteoclasts

Estrogens play a key role in regulation of bone mass and strength by controlling activity of bone‐forming osteoblasts and bone‐resorbing osteoclasts. Cellular effects of estrogens are mediated predominantly by the action of estrogen receptor alpha (ERα). In earlier studies, ablation of the ERα gene in mice did not result in osteoporotic phenotypes due to systemic endocrine disturbance and compensatory effects of elevated levels of testosterone. Despite the relatively well‐established effects in osteoblasts, little is known about the direct action of estrogen in osteoclasts. Development in the last decade of more sophisticated genetic manipulation approaches opened new possibilities to explore cell‐specific roles of nuclear receptors in bone tissue. Recently, we have generated osteoclast‐specific ERα gene knockout mice and shown that in vivo estrogens directly regulate the life span of mature osteoclasts by inducing the expression of pro‐apoptotic Fas ligand (FasL). Inhibitory effects of estrogens on osteoclast function were further studied in vitro. We observed sufficiently detectable ERα expression in osteoclasts differentiating from primary bone marrow cells or RAW264 cells, although levels of ERα were decreasing during progression of the differentiation into mature osteoclasts. Treatment with estrogens led to reduction in expression of osteoclast‐specific genes controlling bone resorption activity. However, estrogens did not affect the size of multinucleated osteoclasts or number of nuclei in a mature osteoclast. In conclusion, in osteoclasts, estrogens function to inhibit bone resorption activity and vitality rather than differentiation.

Minireview: Osteoprotective Action of Estrogens Is Mediated by Osteoclastic Estrogen Receptor-α

The osteoprotective action of estrogen in women has drawn considerable attention because estrogen deficiency-induced osteoporosis became one of the most widely spread diseases in developed countries. In men, the significance of estrogen action for bone health maintenance is also apparent from the osteoporotic phenotype seen in male patients with genetically impaired estrogen signaling. Severe bone loss and high bone turnover, including typical osteofeatures seen in postmenopausal women, can also be recapitulated in rodents after ovariectomy. However, the expected osteoporotic phenotype is not observed in female mice deficient in estrogen receptor (ER)-alpha or -beta or both, even though the degenerative defects are clearly seen in other estrogen target tissues together with up-regulated levels of circulating testosterone. It has also been reported that estrogens may attenuate bone remodeling by cell autonomous suppressive effects on osteoblastogenesis and osteoclastogenesis. Hence, the effects of estrogens in bone appear to be complex, and the molecular role of bone estrogen receptors in osteoprotective estrogen action remains unclear. Instead, it has been proposed that estrogens indirectly control bone remodeling. For example, the enhanced production of cytokines under estrogen deficiency induces bone resorption through stimulation of osteoclastogenesis. However, the osteoporotic phenotype without systemic defects has been recapitulated in female (but not in male) mice by osteoclast-specific ablation of the ERalpha, proving that bone cells represent direct targets for estrogen action. An aberrant accumulation of mature osteoclasts in these female mutants indicates that in females, the inhibitory action of estrogens on bone resorption is mediated by the osteoclastic ERalpha through the shortened lifespan of osteoclasts.

Nuclear Receptors in Bone Physiology and Diseases

During the last decade, our view on the skeleton as a mere solid physical support structure has been transformed, as bone emerged as a dynamic, constantly remodeling tissue with systemic regulatory functions including those of an endocrine organ. Reflecting this remarkable functional complexity, distinct classes of humoral and intracellular regulatory factors have been shown to control vital processes in the bone. Among these regulators, nuclear receptors (NRs) play fundamental roles in bone development, growth, and maintenance. NRs are DNA-binding transcription factors that act as intracellular transducers of the respective ligand signaling pathways through modulation of expression of specific sets of cognate target genes. Aberrant NR signaling caused by receptor or ligand deficiency may profoundly affect bone health and compromise skeletal functions. Ligand dependency of NR action underlies a major strategy of therapeutic intervention to correct aberrant NR signaling, and significant efforts have been made to design novel synthetic NR ligands with enhanced beneficial properties and reduced potential negative side effects. As an example, estrogen deficiency causes bone loss and leads to development of osteoporosis, the most prevalent skeletal disorder in postmenopausal women. Since administration of natural estrogens for the treatment of osteoporosis often associates with undesirable side effects, several synthetic estrogen receptor ligands have been developed with higher therapeutic efficacy and specificity. This review presents current progress in our understanding of the roles of various nuclear receptor-mediated signaling pathways in bone physiology and disease, and in development of advanced NR ligands for treatment of common skeletal disorders.

Structural investigation of the ligand binding domain of the zebrafish VDR in complexes with 1α,25(OH)2D3 and Gemini: purification, crystallization and preliminary X-ray diffraction analysis ☆

The nuclear receptor of Vitamin D can be activated by a large number of agonist molecules with a wide spectrum in their stereochemical framework. Up to now most of our structural information related to the protein-ligand complex formation is based on an engineered ligand binding domain (LBD) of the human receptor. We now have extended our database, using a wild-type LBD from zebrafish that confirms the previously reported results and allows to investigate the binding of ligands that induce significant conformational changes at the protein level.

PPAR-γ Signaling Crosstalk in Mesenchymal Stem Cells

Peroxisome proliferator-activated receptor-gamma (PPAR-γ) is a member of the nuclear receptor (NR) superfamily of ligand-activated transcriptional factors. Among other functions, PPAR-γ acts as a key regulator of the adipogenesis. Since several cytokines (IL-1, TNF-α, TGF-β) had been known to inhibit adipocyte differentiation in mesenchymal stem cells (MSCs), we examined the effect of these cytokines on the transactivation function of PPAR-γ. We found that the TNF-α/IL-1-activated TAK1/TAB1/NIK (NFκB-inducible kinase) signaling cascade inhibited both the adipogenesis and Tro-induced transactivation by PPAR-γ by blocking the receptor binding to the cognate DNA response elements. Furthermore, it has been shown that the noncanonical Wnts are expressed in MSCs and that Wnt-5a was capable to inhibit transactivation by PPAR-γ. Treatment with Wnt5a-activated NLK (nemo-like kinase) induced physical association of the endogenous NLK and H3K9 histone methyltransferase (SETDB1) protein complexes with PPAR-γ. This resulted in histoneH3K9 tri-methylation at PPAR-γ target gene promoters. Overall, our data show that cytokines and noncanonical Wnts play a crucial role in modulation of PPAR-γ regulatory function in its target cells and tissues.

Hormonal gene regulation through DNA methylation and demethylation

Methylation and demethylation of cytosine residues in the genomic DNA play key roles in a wide range of fundamental biological processes such as differentiation and development, genome stability, imprinting, X chromosome inactivation, carcinogenesis and aging. DNA methylation is considered to be a stable modification associated with the epigenetic silencing of genomic loci and maintained through cellular division. Recent studies however, suggest that DNA methylation and demethylation are considerably more dynamic than previously thought and may be involved in repression and derepression of gene activity during the lifespan of a cell. This article is focused on epigenetic mechanisms in the hormonal regulation of the cytochrome p450 27B1 or CYP27B1 gene activity that involve reversible epigenetic modifications to chromatin and DNA methylation profiles.