László G. Kömüves

HOXA9 Forms Triple Complexes with PBX2 and MEIS1 in Myeloid Cells

ABSTRACT Aberrant activation of the HOX, MEIS, and PBX homeodomain protein families is associated with leukemias, and retrovirally driven coexpression of HOXA9 and MEIS1 is sufficient to induce myeloid leukemia in mice. Previous studies have demonstrated that HOX-9 and HOX-10 paralog proteins are unique among HOX homeodomain proteins in their capacity to form in vitro cooperative DNA binding complexes with either the PBX or MEIS protein. Furthermore, PBX and MEIS proteins have been shown to form in vivo heterodimeric DNA binding complexes with each other. We now show that in vitro DNA site selection for MEIS1 in the presence of HOXA9 and PBX yields a consensus PBX-HOXA9 site. MEIS1 enhances in vitro HOXA9-PBX protein complex formation in the absence of DNA and forms a trimeric electrophoretic mobility shift assay (EMSA) complex with these proteins on an oligonucleotide containing a PBX-HOXA9 site. Myeloid cell nuclear extracts produce EMSA complexes which appear to contain HOXA9, PBX2, and MEIS1, while immunoprecipitation of HOXA9 from these extracts results in coprecipitation of PBX2 and MEIS1. In myeloid cells, HOXA9, MEIS1, and PBX2 are all strongly expressed in the nucleus, where a portion of their signals are colocalized within nuclear speckles. However, cotransfection of HOXA9 and PBX2 with or without MEIS1 minimally influences transcription of a reporter gene containing multiple PBX-HOXA9 binding sites. Taken together, these data suggest that in myeloid leukemia cells MEIS1 forms trimeric complexes with PBX and HOXA9, which in turn can bind to consensus PBX-HOXA9 DNA targets.

EXPRESSION AND SIGNAL TRANSDUCTION OF CALCIUM-SENSING RECEPTORS IN CARTILAGE AND BONE

We previously showed that Ca 21 -sensing receptors (CaRs) are expressed in chondrogenic RCJ3.1C5.18 (C5.18) cells and that changes in extracellular [Ca 21 ] ([Ca 21 ]o) modulate nodule formation and chondrogenic gene expression. In the present study, we detected expression of CaRs in mouse, rat, and bovine cartilage and bone by in situ hybridization, immunocytochemistry, immunoblotting, and RT-PCR; and we tested the effects of CaR agonists on signal transduction in chondrogenic and osteogenic cell lines. In situ hybridization detected CaR transcripts in most articular chondrocytes and in the hypertrophic chondrocytes of the epiphyseal growth plate. Expression of CaR transcripts was weak or absent, however, in proliferating and maturing chondrocytes in the growth plate. In bone, CaR transcripts were present in osteoblasts, osteocytes, and bone marrow cells, but rarely in osteoclasts. A complementary DNA was amplified from mouse growth plate cartilage, which was highly homologous to the human parathyroid CaR sequence. Immunocytochemistry of cartilage and bone with CaR antisera confirmed these findings. Western blotting revealed specific bands (;140 ‐190 kDa) in membrane fractions isolated from growth plate cartilage, primary cultures of rat chondrocytes, and several osteogenic cell lines (SaOS-2, UMR-106, ROS 17/2.8, and MC3T3-E1). InsP responses to high [Ca 21 ]o were evident in C5.18 cells and all osteogenic cell lines tested except for SaOS-2 cells. In the latter, high [Ca 21 ]o reduced PTH-induced cAMP formation. Raising [Ca 21 ]o also increased intracellular free [Ca 21 ]i n SaOS-2 and C5.18 cells. These studies confirm expression of CaRs in cartilage and bone and support the concept that changes in [Ca 21 ]o may couple to signaling pathways important in skeletal metabolism. (Endocrinology 140: 5883‐5893, 1999)

The Calcium Sensing Receptor and Its Alternatively Spliced Form in Murine Epidermal Differentiation

We have recently reported that human keratinocytes express both the full-length calcium sensing receptor (CaR) and an alternatively spliced form lacking exon 5, which were suggested to be involved in calcium induced keratinocyte differentiation. To understand further the role of these CaRs, we analyzed the structure of mouse CaRs, and investigated their role using a mouse model in which only the full-length CaR was disrupted. Our results show that both the full-length and the alternatively spliced variant lacking exon 5 encoding 77 amino acids of the extracellular domain were expressed in mouse epidermis. The deletion of the full-length CaR increased the production of the alternatively spliced form of CaR in mutant mice. The keratinocytes derived from these mutant mice did not respond to extracellular calcium, suggesting that the full-length CaR is required to mediate calcium signaling in the keratinocytes. The loss of the full-length CaR altered the morphologic appearance of the epidermis and resulted in a reduction of the mRNA and protein levels of the keratinocyte differentiation marker, loricrin. These results indicate that CaR is important in epidermal differentiation, and that the alternatively spliced form does not fully compensate for loss of the full-length CaR.

Epidermal expression of the full-length extracellular calcium-sensing receptor is required for normal keratinocyte differentiation

The importance of the extracellular calcium‐sensing receptor (CaR) in the stringent control of extracellular Ca2+ concentration is well established. However, the presence of CaR in tissues not directly involved in regulating mineral ion homeostasis such as the epidermis suggests a role for CaR in other cellular functions. Although extracellular Ca2+ regulates the differentiation of epidermal keratinocytes, the role of CaR in this process in the epidermis is not fully understood. In this study we showed using in situ hybridization and immunohistochemistry that CaR is expressed in suprabasal keratinocytes of the mammalian epidermis. We then evaluated the changes in epidermal keratinocyte morphology and differentiation in Casr−/− mice lacking the full‐length CaR. These mice show increased expression of an alternatively spliced form of CaR which lacks acute Ca2+‐signaling properties. The absence of the full‐length CaR in the epidermis resulted in ultrastructural changes (abnormal keratohyalin granule formation and precocious lamellar body secretion) in the terminally differentiated granular keratinocytes. Furthermore, the expression of both mRNA and protein for the calcium inducible keratinocyte differentiation markers, filaggrin and loricrin, were down‐regulated in the epidermis of Casr−/− mice, whereas the number of proliferating cells were increased even though the calcium gradient within the epidermis was enhanced. Our results demonstrate that the epidermal expression of the full‐length CaR is required for the normal terminal differentiation of keratinocytes.

Changes in HOXB6 homeodomain protein structure and localization during human epidermal development and differentiation

HOX homeodomain proteins are master developmental regulators, which are now thought to function as transcription factors by forming cooperative DNA binding complexes with PBX or other protein partners. Although PBX proteins exhibit regulated subcellular localization and function in the nucleus in other tissues, little data exists on HOX and PBX protein localization during skin development. We now show that the HOXB6 protein is expressed in the suprabasal layer of the early developing epidermis and throughout the upper layers of late fetal and adult human skin. HOXB6 signal is cytoplasmic throughout fetal epidermal development, but substantially nuclear in normal adult skin. HOXB6 protein is also partially nuclear in hyperproliferative skin conditions, but appears to be cytoplasmic in basal and squamous cell carcinomas. Although all three PBX genes are expressed in fetal epidermis, none of the three PBX proteins exhibit nuclear co‐localization with HOXB6 in either fetal or adult epidermis. RNA and protein data suggest that a truncated HOXB6 protein, lacking the homeodomain, is expressed in undifferentiated keratinocytes and that the full‐length protein is induced by differentiation. GFP‐fusion proteins were used to demonstrate that the full‐length HOXB6 protein is localized to the nucleus while the truncated protein is largely cytoplasmic. Taken together, these data suggest that during epidermal development the truncated HOXB6 isoform may function by a mechanism other than as DNA binding protein, and that most of the nuclear, homeodomain‐containing HOXB6 protein does not utilize PBX proteins as DNA binding partners in the skin. Published 2000 Wiley‐Liss, Inc.

HOXB4 homeodomain protein is expressed in developing epidermis and skin disorders and modulates keratinocyte proliferation

The HOX homeodomain proteins are fundamental regulators of organ and tissue development, where they are thought to function as transcription factors, and HOX gene expression has been associated with numerous types of cancers. Previous studies have demonstrated that enforced expression of the HOXB4 protein transforms cultured fibroblasts and leads to a selective expansion of the hematopoietic stem cell pool, suggesting that this protein might play a role in cellular proliferation. In support of this concept, we now show that enforced expression of HOXB4 in human neonatal keratinocytes results in increased cellular proliferation and colony formation as well as decreased expression of the alpha‐2‐integrin and CD44 cell surface adhesion molecules. We previously have reported HOXB4 gene expression in the basal and suprabasal layers of developing human skin and now show extensive HOXB4 mRNA in psoriatic skin and basal cell carcinoma. In fetal human skin HOXB4 protein expression was both nuclear and cytoplasmic within epidermal basal cells and in hair follicle inner and outer root sheath cells, whereas strong nuclear signals were observed in the bulge region. In adult skin, HOXB4 protein expression was both nuclear and cytoplasmic, but was predominantly localized to the intermediate and differentiated cell layers. In contrast to the striking gradient patterns of HOX gene and protein expression previously described in developing spinal cord and limb, HOXB4 protein was uniformly detected in all regions of the fetal and adult skin. Although little HOXB4 signal localized to proliferative cell layers, as marked by proliferating cell nuclear antigen (PCNA) staining, in normal adult epidermis, nuclear HOXB4 protein expression substantially overlapped with PCNA‐positive cell in a series of samples of hyperproliferative skin. Taken together, these data suggest that nuclear HOXB4 protein may play a role in the regulation of cellular proliferation/adhesion in developing fetal human epidermis and in hyperproliferation conditions, including cancers, in adult epidermis. Published 2002 Wiley‐Liss, Inc.

HOXB13 homeodomain protein is cytoplasmic throughout fetal skin development

Substantial evidence suggests that HOX homeobox genes regulate aspects of body development, including hair formation. We initially isolated the HOXB13 gene from human fetal skin in experiments designed to identify candidate genes that regulate scarless fetal wound healing. Although the HOX homeodomain proteins have been proposed to function as transcription factors, we have demonstrated previously that substantial fractions of the HOXB6 and HOXB4 proteins are localized to the cytoplasm throughout epidermal development. The purpose of the current study was to identify HOXB13 protein expression patterns in developing skin to elucidate potential mechanisms by which this protein might regulate aspects of tissue development and healing. HOXB13 protein expression was detected throughout the developing epidermis, with weaker signal observed in the early developing dermis. Epidermal HOXB13 signal was detected over the entire body surface, but surprisingly, essentially all of the signal was cytoplasmic in developing skin. Low‐level HOXB13 protein expression was detected in adult skin and within the telogen hair follicle, and a portion of the residual signal in adult epidermis was nuclear. Expression in hyperproliferative skin conditions remained cytoplasmic with the exception of epidermis associated with Kaposis sarcoma, which showed strong HOXB13 expression that was partially localized to the nucleus. Developmental Dynamics 227:192–202, 2003.

ZIPPER PROTEIN, A B-G PROTEIN WITH THE ABILITY TO REGULATE ACTIN/MYOSIN 1 INTERACTIONS IN THE INTESTINAL BRUSH BORDER

We recently identified a 28-kDa protein in the intestinal brush border that resembled tropomyosin in terms of size, homology, and α helical content. This protein contained 27 heptad repeats, nearly all of which began with leucine, leading to its name zipper protein. Subsequent analysis, however, indicated that both a 49-kDa and a 28-kDa immunoreactive protein existed in intestinal brush-border extracts. Using 5′-rapid amplification of cDNA ends analysis, we extended the N-terminal sequence of zipper protein to the apparent translation start site. This additional sequence contained a putative transmembrane domain and two potential tryptic cleavage sites C-terminal to the transmembrane domain which would release a 28-kDa cytoplasmic protein if utilized. The additional sequence was highly homologous to members of the B-G protein family, a family with no known function. Immunoelectron microscopy showed that zipper protein was confined to the membrane of the microvillus where it was in close association with brush-border myosin 1 (BBM1). Recombinant zipper protein (28-kDa cytoplasmic portion) blocked the binding of actin to BBM1 and inhibited actin-stimulated BBM1 ATPase activity. In contrast, zipper protein had no effect on endogenous or K/EDTA-stimulated BBM1 ATPase activity. Furthermore, zipper protein displaced tropomyosin from binding to actin, suggesting that these homologous proteins bind to the same sites on the actin molecule. We conclude that zipper protein is a transmembrane protein of the B-G family localized to the intestinal epithelial cell microvillus. The extended cytoplasmic tail either in the intact molecule or after tryptic cleavage may participate in regulating the binding and, thus, activation of BBM1 by actin in a manner similar to tropomyosin.

Analysis of HOX homeodomain proteins and gene transcripts in the epidermis.

HOX homeodomain proteins are thought to be master developmental regulators of tissue patterning during embryogenesis. These DNA binding proteins also have diverse roles in adult cell function, and derangement of HOX genes has been associated with several types of cancer. In this chapter we present protocols for the immunohistochemical localization of HOX proteins in the epidermis. We also provide in situ hybridization protocols for detection of HOX gene mRNA transcripts in the epidermis.