Role of Gadd45 in Myeloid Cells in Response to Hematopoietic Stress

Abstract

The gadd45 family of genes is rapidly induced by different stressors, including differentiation-inducing cytokines, and there is a large body of evidence that their cognate proteins are key players in cellular stress responses. Induction of gadd45 genes at the onset of myeloid differentiation suggested that Gadd45 protein(s) play a role in hematopoiesis, yet no apparent abnormalities were observed in either the bone marrow or peripheral blood compartments of mice deficient for either gadd45a or gadd45b. However, under conditions of hematological stress, including acute stimulation with cytokines, myelo-ablation and inflammation, both gadd45a-deficient and gadd45b-deficient mice exhibited deficiencies. This topic is discussed within the context of what is known about Gadd45 proteins in stress signaling, hematopoietic development and the innate immune response.

Introduction

The gadd45 family of genes encodes for small (18kd), evolutionarily conserved proteins that are highly homologous to each other (55%–57% overall identity at the amino acid level), are highly acidic, and are primarily, but not exclusively, localized within the cell nucleus. [1–4]. These genes are rapidly induced by different stressors, including differentiation-inducing cytokines, and there is a large body of evidence that their cognate proteins are key players in cellular stress responses.

Gadd45a, originally termed gadd45, was first cloned as one of many gadd (growth arrest and DNA damage-inducible) genes on the basis of rapid induction by UV radiation in Chinese hamster ovary (CHO) cells [1, 2]. Gadd45b, originally termed MyD118, was first cloned as a myeloid differentiation primary response gene, induced in the absence of protein synthesis following treatment of M1 myeloblastic leukemia cells with differentiation inducers [1, 5]. Gadd45g, isolated using an MyD118 cDNA fragment encoding for amino acids 37–92, which is about 80% homologous to gadd45, to screen for other potential members of the gadd45 gene family, was subsequently found to encode for the murine homologue of human CR6 [3]. CR6 was originally cloned as an immediate early response gene in T cells stimulated by interleukin-2 [6].

Each of these genes is expressed in multiple murine tissues including heart, brain, spleen, lung, liver, skeletal muscle, kidney and testes, but at different levels [3]. Furthermore, expression of gadd45a, gadd45b and gadd45g is induced in response to multiple environmental and physiological stresses, including MMS, IR, UV, VP-16, daunorubicin (DNR), and inflammatory cytokines [1, 7–8]. In all cases the pattern of expression for each gadd45 gene is unique, consistent with each gadd45 family member playing a different role in response to each source of stress. In addition it was observed that during myeloid differentiation, using either normal bone marrow (BM) stimulated with different hematopoietic cytokines or various hematopoietic cell lines induced to undergo terminal myeloid differentiation, each gadd45 gene has a distinctive pattern of expression [3, 8]. Consistent with the distinctive expression patterns, regulation of expression for each gadd45 gene differs. For instance, Gadd45a is a p53 target gene, although its inductions can also be p53 independent, whereas gadd45b is induced by both IL-6 and TGF-β in the absence of de novo protein synthesis and gadd45g is induced as a primary response to both IL-2 and IL-6 [3, 4, 6, 9].

Gadd45 proteins are implicated in cycle arrest [10–13], DNA repair [14–17], cell survival and apoptosis [7, 9, 18–25] in response to environmental and physiological stress, and display a complex array of physical interactions with other cellular proteins; these protein-protein interactions are implicated in cell cycle regulation and the response of cells to stress. Proteins that interact with Gadd45 proteins include PCNA, cdk1, p21, MEKK4 and p38, where interaction with MEKK4 and p38 results in their activation and interaction with cdk1 inhibits the kinase activity of the cdc2/cyclinB1 complex [11, 13, 14, 16, 19, 20]. It is possible that the nature of the stress stimulus encountered, its magnitude, and the cell type regulate the interaction of Gadd45 proteins with other proteins that modulate Gadd45 function. It is expected that, in addition to other variables, specific protein-protein interactions are regulated by the level of expression, cellular localization, and posttranslational modifications of both the Gadd45 proteins and their interacting partners. Which partner each Gadd45 protein associates with within a specific biological setting will ultimately determine the specific biochemical function for each Gadd45 protein. This knowledge should give a better understanding of how the varied functions of the Gadd45 proteins are manifested.

Expression of gadd45 in myeloid cells in response to cytokine stimulation

Using the M1 myeloid leukemic cell line, which is a model for terminal myeloid differentiation segregated from proliferation, this laboratory has shown that gadd45b is a differentiation primary response gene induced immediately and then down-regulated, gadd45g is also induced immediately and not dependent on de novo protein sysnthesis, however its expression peaks only at one day and continues to be expressed throughout the differentiation program. Finally, gadd45a is induced only at later times. Myeloid enriched BM treated with IL-3, GM-CSF, G-CSF or M-CSF results in rapid induction of all three gadd45 genes, yet the patterns of expression vary during the ensuing myeloid developmental program [3, 5, 8]. The expression of gadd45b in gadd45a−/− BM and of gadd45a in gadd45b−/− BM was comparable to their expression in wild type BM cells. Gadd45g expression was the same for all genotypes. These expression studies indicate that gadd45 genes are independently induced in response to hematopoietic cytokines.

Induction of gadd45 genes at the onset of myeloid differentiation suggested that Gadd45 protein(s) play a role in myelopoiesis, yet no apparent abnormalities were observed in the number and types of hematopoietic cells in either the BM or peripheral blood compartments of 2 months old mice deficient for either gadd45a or gadd45b [8]. Since the gadd45 gene products are known stress sensors, myeloid cells from gadd45a−/− and gadd45b−/− mice were examined under conditions of hematopoietic stress, both in vivo and in vitro.

Role of gadd45 in the response of myeloid cells to acute stimulation with cytokines

After 4 days of acute stimulation of myeloid enriched BM with hematopoietic cytokines GM-CSF, IL-3, M-CSF, or G-CSF, the percentage of mature macrophages and granulocytes was significantly reduced in gadd45a−/− and gadd45b−/− BM cells compared to wild type controls, using both morphological and immuno-phenotyping [8]. Gadd45a−/− and gadd45b−/− BM cells grown in IL-3 or GM-CSF did not differ significantly from wild type cells with regard to apoptosis; however, significantly increased apoptosis was observed in gadd45a−/− and gadd45b−/− BM cells following treatment with either M-CSF or G-CSF compared to wild type controls [8]; this included both differentiated and undifferentiated myeloid cells, indicating that gadd45a and gadd45b are important modulators of cell survival in both undifferentiated and differentiated myeloid cells upon acute stimulation with either M-CSF or G-CSF. Furthermore, the effect of gadd45 deficiency on the clonogenic potential of myeloid progenitor cells consisted of an initial decrease in colony formation with a subsequent increase in clonability of colony forming units granulocyte/macrophage progenitors [8]. These observations, indicative of prolonged proliferation capacity and decreased ability to differentiate, reflect the compromised cytokine induced myeloid differentiation and enhanced apoptosis.

It is known that signaling pathways emerging from cytokine receptors activate tyrosine kinases, which phosphorylate substrates recruited to the receptor complex, including Stat3 and Stat5. Phosphorylated Stat3 and Stat5 translocate to the nucleus where they activate the transcription of genes associated with cell survival, proliferation and differentiation [26, 27], including the gadd45b and gadd45g genes that are primary responders [5, 6]. Consistent with this notion is the observed loss of gadd45 induction in splenocytes deficient for either Stat5 or Jak3 after anti-CD3 stimulation [28]. Hematopoietic cytokines also activate the MAP kinases including p38, JNK and ERK [29]. There is ample evidence that the different Gadd45 proteins are modulators of these signaling pathways, and that deficiency in Gadd45 proteins alters the signaling, and ultimately stress induced myelopoiesis in gadd45 deficient mice [18, 22, 23, 30].

Role of gadd45 in maintaining the quiescent stem cell pool

Administration of the anti-metabolite 5-FU results in ablation of proliferating progenitor cells in BM, and hence myelo-suppression. To recover, quiescent cells from the stem cell pool are activated to replenish the progenitor pool, which subsequently differentiates along multiple cell lineages [31]. Under these conditions, the cellularity of the BM in gadd45a−/− and gadd45b−/− mice appeared to be significantly reduced and displayed poor recovery of the mature myeloid compartment compared to wild type controls [8]. These observations indicate that following acute myeloid suppression, deficiencies in either gadd45a or gadd45b prolong the time needed for recovery of the myeloid compartment. Several explanations for these observations are available that merit further investigation, and it is also possible that more than one is in play to determine the ultimate outcome following myelo-ablation. It can be asked if gadd45 gene products directly modulate the stem cell compartment, co-operating in a stage specific function that may be related to cell survival and/or self-renewal, so that loss of gadd45 diminishes the stem cell population of quiescent cells. Alternatively, the poor recovery of the mature myeloid compartment following myelo-ablation can reflect a defect in terminal myeloid differentiation and diminished survival. Another possibility is that diminished survival of progenitors accelerates depletion of the stem cell compartment. It is of definite interest to assess the possible role of Gadd45 protein interaction with p21, one of its partner proteins (16), in the above described defect in myeloid recovery, since p21 has been implicated in the regulation of hematopoietic stem cell quiescence and cell survival [32, 33].

The role of gadd45 in the response of the myeloid compartment to acute inflammatory stress

Intraperitoneal delivery of sodium caseinate rapidly induces myelopoiesis in murine BM and also results in prompt migration of inflammatory cells from the BM and other hematopoietic reservoirs to the peritoneal cavity, where there is an increase in mature myeloid cells (& lymphocytes) 3–4 days after sodium caseinate injection. BM cells of wild type mice subjected to sodium caseinate treatment consisted mostly of Gr-1 positive myeloid cells (98.6%) compared to 76.4% in untreated mice; however, there was no substantial difference in the proportion of Gr-1 positive BM cells in either gadd45a−/− or gadd45b−/− mice compared to untreated controls [8]. Peritoneal exudates from gadd45a deficient and gadd45b deficient mice contain significantly fewer cells and the percentage of both F4/80 and Gr-1 expressing cells was appreciably lower compared to peritoneal exudates of similarly treated wild type mice, indicating that both Gadd45a and Gadd45b are important modulators of the response of myeloid cells to acute inflammatory stress [8].

Loss of either gadd45a or gadd45b compromises the innate immune function of the myeloid compartment. This defect can be due to impaired differentiation, migration and functions of leukocytes in response to inflammatory stress, among many possibilities. Whether this is the case, and how it integrates with the effect of loss of gadd45 function in the lymphoid [34] or non-hematopoietic cell compartments, including epithelial cells, needs to be explored. Recent evidence has implicated Gadd45 proteins in T cell functions related to innate immunity [34–36] and regulation of dendritic cell cytokine production [37], where Gadd45 regulation of the stress-activated MAP kinase pathways has been implicated in these findings, and have also been shown to be required for the response of myeloid cells to genotoxic stress [30, 34–37].

Concluding remarks

The mechanisms that underlie the function of Gadd45 proteins in modulating the response of myeloid cells to different types of hematopoietic stress are being investigated. In addition to using mice deficient for each gadd45 gene, mice deficient for two gadd45 genes will be analyzed. Genetic lesions altering expression or function of any of the gadd45 gene products may impinge on the ability of an organism to respond to any challenge to the immune system. Individuals may have altered responses to infection and/or toxic shock, which may be further exacerbated in elderly individuals since older mice deficient in either gadd45a or gadd45b exhibit cytopenia in their peripheral blood. Similarly, individuals with defects in gadd45 may be more adversely affected compared to normal individuals by treatments that stress the hematopoietic stem cell compartment, including anti-cancer chemotherapeutic agents. Integrating molecular, cellular and genetic studies with animal models should further clarify the involvement of gadd45 in the different hematopoietic compartments following hematopoietic stress.