Disease relevance of HOXB4

• When injected separately into nonobese diabetic-severe combined immunodeficient (NOD/SCID) mice or in competition with control vector-transduced cells, HOXB4-overexpressing cord blood CD34+ cells had a selective growth advantage in vivo, which resulted in a marked enhancement of the primitive CD34+ subpopulation (P =.01) [1].
• HOXA5, but not HOXB4, -B5, or -B7 activated the PR promoter in two breast cancer cell lines, MCF-7 and Hs578T [2].
• 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 [3].
• To circumvent the requirement for retroviral infection, we used recombinant human TAT-HOXB4 protein carrying the protein transduction domain of the HIV transactivating protein (TAT) as a potential growth factor for stem cells [4].
• HSCs exposed to TAT-HOXB4 for 4 d expanded by about four- to sixfold and were 8-20 times more numerous than HSCs in control cultures, indicating that HSC expansion induced by TAT-HOXB4 was comparable to that induced by the human HOXB4 retrovirus during a similar period of observation [4].

High impact information on HOXB4

• Ex vivo expansion of human hematopoietic stem cells by direct delivery of the HOXB4 homeoprotein [5].
• Here we show that when cultured on stromal cells genetically engineered to secrete HOXB4, human long-term culture-initiating cells (LTC-ICs) and nonobese diabetic-severe combined immunodeficiency (NOD-SCID) mouse repopulating cells (SRCs) were expanded by more than 20- and 2.5-fold, respectively, over their input numbers [5].
• To eliminate any deleterious effects that might be associated with stable HOXB4 gene transfer into human cells, we took advantage of the ability of HOX proteins to passively translocate through cell membranes [5].
• Retroviral overexpression of the human homeobox B4 (HOXB4) gene in mouse bone marrow cells enables over 40-fold expansion of HSCs in vitro [4].
• To assess the role these genes may play in regulating the proliferation and/or differentiation of such cells, we engineered the overexpression of HOXB4 in murine bone marrow cells by retroviral gene transfer and analyzed subsequent effects on the behavior of various hematopoietic stem and progenitor cell populations both in vitro and in vivo [6].

Biological context of HOXB4

• Cellular proliferation and transformation induced by HOXB4 and HOXB3 proteins involves cooperation with PBX1 [7].
• In situ hybridization analysis also revealed strong HOXC4 and weaker HOXB7 expression throughout fetal development, whereas HOXB4 was expressed at barely detectable levels [8].
• HOXB4 expression in K562 cells is regulated at the level of transcription, and transient transfection defines primary HOXB4 regulatory sequences within a 99-bp 5' promoter [9].
• Mutations within the HOXB4 promoter identified a potential E box binding site (HOX response element [HXRE]-2) as the most critical regulatory sequence, and yeast one hybrid assays evaluating bone marrow and K562 libraries for HXRE-2 interaction identified upstream stimulating factor (USF)-2 and micropthalmia transcription factor (MITF) [9].
• The data further establish that HOXB4 levels are regulated by 1,25-(OH)2D(3) and reveal that HOXB4 participates in the down-regulation of c-myc expression [10].

Anatomical context of HOXB4

• We have recently shown that certain members of the Hox A and B clusters, such as HOXB3 and HOXB4, are preferentially expressed in subpopulations of human bone marrow that are highly enriched for the most primitive hematopoietic cell types [6].
• High-level ectopic HOXB4 expression confers a profound in vivo competitive growth advantage on human cord blood CD34+ cells, but impairs lymphomyeloid differentiation [1].
• Furthermore, HOXB4 overexpression also significantly reduced B-cell output (P <.01) [1].
• HOXB4 homeodomain protein is expressed in developing epidermis and skin disorders and modulates keratinocyte proliferation [3].
• 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 [3].

Associations of HOXB4 with chemical compounds

• In this study, a phosphorothioate antisense oligonucleotide targeted against HOXB4 was examined for its effect on 1,25-(OH)2D(3) inhibition of c-myc expression [10].
• In addition, HOXB4 antisense partially blocked 1,25-(OH)2D(3)-mediated decrease in c-myc levels (46+/-6% inhibition) and promotion of HL-60 cell differentiation (20+/-2% and 25+/-3% inhibition as assessed by nitroblue tetrazolium and non-specific esterase assays respectively) [10].
• Recently, it has been shown that expression of human HOX 2 genes is sequentially activated by RA beginning from Hox 2.9 at the 3' end of the HOX 2 cluster (A. Simeone, D. Acampora, L. Arcioni, P. W. Andrews, E. Boncinelli, and F. Mavilio, Nature [London] 346:763-766, 1990) [11].
• Moreover, valproic acid treatment increases histone H4 acetylation levels at specific regulatory sites on HOXB4, a transcription factor gene with a key role in the regulation of HSC self-renewal and AC133, a recognized marker gene for stem cell populations [12].
• HOXB4 levels were significantly increased in response to 1,25-(OH)2D3 [13].

Regulatory relationships of HOXB4

• Culture of highly purified human CD34(+) bone marrow cells in thrombopoietin/Flt-3 ligand/stem cell factor induced HOXB4 3-10-fold, whereas culture in granulocyte/macrophage colony-stimulating factor, only increased HOXB4/luciferase expression 20-50% [9].
• Deregulated expression of HOXB4 enhances the primitive growth activity of human hematopoietic cells [14].
• Furthermore, Smad1 inhibited Hoxb4-mediated target gene Irx5 expression during early Xenopus development [15].

Other interactions of HOXB4

• 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 [16].
• To explore if these patterns reflect different functional activities, we have retrovirally engineered the overexpression of a 5'-located gene, HOXA10, in murine bone marrow cells and demonstrate effects strikingly different from those induced by overexpression of a 3'-located gene, HOXB4 [17].
• Overexpression of either HOXB4 or HOXA9 in primitive marrow cells enhances the expansion of hematopoietic stem cells (HSCs) [18].
• 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 [3].
• Our preliminary results indicate that the expanding potential of HOXB4 is retained and even augmented by fusion to NUP98 [19].

Analytical, diagnostic and therapeutic context of HOXB4

• Electrophoretic mobility shift assay with K562 extracts confirmed that these proteins, along with USF-1, bind to the HOXB4 promoter in vitro [9].
• Most important, NF-Ya subunit protein levels are found to be lower in c-Kit-Gr-1+ granulocytic bone marrow (BM) cells than in c-Kit+ immature BM cells, in parallel with a reduction of NF-Y occupancy on the HOXB4 promoter as shown by chromatin immunoprecipitation (ChIP) assay [20].
• These results show for the first time unwanted side effects of ectopic HOXB4 expression and therefore underscore the need to carefully determine the therapeutic window of HOXB4 expression levels before initializing clinical trials [1].
• The most dramatic effect of HOXB4 was observed early after transplantation, resulting in an up to 56-fold higher engraftment compared to the control cells [21].
• We have also investigated the region-specific expression of HOX-2 genes in human embryonic-fetal life by Northern-blot analysis [22].

References