CCAAT/enhancer-binding protein-β has a role in osteoblast proliferation and differentiation

Abstract

CCAAT/enhancer binding protein β (C/EBPβ) is known to play an important role in the expression of several genes necessary for bone development and homeostasis including osteocalcin, IGF-1, and IL-6. In this study, we show that C/EBPβ protein levels and, consequently, DNA-binding activity are temporally regulated, dramatically decreasing upon differentiation of MC3T3-E1 mouse osteoblasts. Corresponding with these results, the constitutive expression of C/EBPβ LAP in MC3T3-E1 osteoblasts increased proliferation and suppressed osteogenic differentiation. Thus, C/EBPβ LAP not only appears to participate in the regulation of genes associated with mature bone physiology, but is also a critical regulator of osteoblast growth and differentiation.

Introduction

CCAAT/enhancer binding protein (C/EBP) α, β, γ, δ, ε, and ζ comprise a family of basic region-leucine zipper (bZIP) transcription factors (reviewed in Ref. [1]). These proteins dimerize through their leucine zipper domains and bind to DNA through their adjacent basic regions. C/EBPα, β, δ, and ε can activate in vivo transcription from promoters that contain a consensus binding site: 5′-T(T/G)NNGNAA(T/G)-3′ [2]. The in vitro functions of these family members are nearly identical to those of prototypical C/EBP, but the variety of C/EBP family members and their potential for heterodimer formation could provide a large repertoire of transcription factors with complex in vivo regulatory features.

C/EBPβ has been most extensively characterized as an effector in the induction of acute phase and inflammatory genes responsive to LPS, IL-1, or IL-6 ([2], [3], [4], [5], reviewed in Ref. [6]). Consistent with this role, the DNA-binding activity of C/EBPβ is increased by a post-transcriptional mechanism when cells are exposed to LPS, IL-1, or IL-6 [2], [5] and C/EBPβ expression is capable of enhancing transcription from promoters containing elements responsive to those factors [2], [3], [4], [5]. Although the primary mechanism of C/EBPβ regulation within inflammatory responses appears to be post-transcriptional [7], C/EBPβ mRNA levels are also induced by LPS, IL-1, or IL-6 [2]. Thus, there is a circular aspect to C/EBPβ regulation with regulated gene products (e.g. IL-1 and IL-6) regulating expression of the regulator (i.e. C/EBPβ).

Among the post-transcriptional mechanisms for C/EBPβ regulation, covalent modification has been shown to be an important mechanism for modulating its activity. Site-specific phosphorylation through the MAP kinase pathway concomitant with increased C/EBPβ transactivation is stimulated by the ras oncogene [8], as well as stimulation by interferon γ [9] and growth hormone [10]. C/EBPβ activity is also stimulated by site-specific phosphorylation in response to elevated intracellular levels of calcium through a calcium/calmodulin-dependent kinase [11]. TPA, acting through the protein kinase C pathway, also increases C/EBPβ site-specific phosphorylation and activity [12]. This phosphorylation is critical for TGFα-induced hepatocyte proliferation [13]. In vitro studies have identified several protein kinase A and C phosphorylation sites that modulate DNA binding, including one that attenuates DNA binding [14], as does site-specific glycogen synthase kinase 3 phosphorylation [15]. All of these instances of modulation of C/EBPβ activity and phosphorylation involve several phosphorylation sites and protein kinases. Clearly, C/EBPβ can be regulated by a variety of signal transduction pathways, perhaps depending upon the cell type in which it is expressed. More recently, C/EBPβ activity has been reported to be increased by sumoylation [16] and to be attenuated by acetylation [17].

Another level of post-transcriptional regulation for C/EBPβ is provided by the expression of three C/EBPβ isoforms, LAP* (38 kDa), LAP (35 kDa), and LIP (20 kDa), through the use of alternative translational start sites in the C/EBPβ mRNA [18] or proteolysis [19], [20]. The regulation of alternative translational initiation depends upon an evolutionarily conserved upstream open reading frame and the regulation of translation factor eIF activity [21], as well as the CUG-repeat binding protein interactions with the 5′ region of the C/EBPβ mRNA [22]. LAP* and LAP both retain three transcriptional activation domain modules, whereas LIP lacks these activation domain modules [23]. LIP is unable to activate transcription from a C/EBP-dependent promoter-reporter and can inhibit C/EBPβ-mediated transcriptional activation [18]. On the other hand, LIP can activate transcription from the IL-6 promoter with LPS stimulation [24]. Overexpression of LIP has been observed in fetal liver cells [18] and in mammary tumors [25] where it is proposed to inhibit the expression of C/EBP-dependent genes that promote differentiation and inhibit proliferation, thus promoting proliferation and/or a transformed phenotype. LAP* differs from LAP by a short N-terminal extension (21 amino acids in rodents) that plays a role in the recruitment of the ATP-dependent SWI/SNF chromatin remodeling complex [26] and in redox regulation of C/EBPβ activity [27]. The fact that LAP* has been observed to be expressed in normal human mammary epithelial cells and absent in cancerous mammary epithelial cells although LAP shows exactly the opposite distribution suggests functional consequences for the differential expression of these two C/EBPβ isoforms [28].

C/EBPβ has been characterized as a regulator of differentiation in several systems. C/EBPβ acts in the cascade of events leading to adipocyte differentiation [29], [30]. C/EBPβ has also been shown to have critical roles in the differentiation of the female reproductive system and mammary gland. Female C/EBPβ-deficient mice are sterile due to a block in granulosa cell differentiation [31] and are defective in mammary gland development [32], [33]. Interestingly, pancreatic cells transdifferentiate into hepatocytes when C/EBPβ is overexpressed [34].

Although these studies suggest roles for C/EBPβ in differentiation, C/EBPβ has been associated with proliferation in two recent studies. C/EBPβ augments Ha-ras-induced transformation of NIH3T3 cells and has a critical role in ras-mediated tumorigenesis of keratinocytes [35]. C/EBPβ can also transform a normal mammary epithelial cell line, conferring properties normally associated with neoplastic cells [36].

A growing literature suggests important roles for C/EBP family members in growth and differentiation of osteoblasts. In primary rat osteoblast cultures, C/EBPβ and C/EBPδ are abundantly expressed in proliferating cells, decline during the mid-stage of differentiation, and then subsequently increase at late stages of differentiation [37]. Both C/EBPβ and C/EBPδ are implicated in the regulation of IGF-1 [38] and osteocalcin [37] in osteoblasts. IGF-1 acts as a growth and differentiation factor for bone (reviewed in Ref. [39]), although osteocalcin expression is associated with fully differentiated osteoblasts (reviewed in Ref. [40]). C/EBPβ and C/EBPδ can also act synergistically with Runx2/Cbfa1, a transcription factor and nuclear matrix binding protein (reviewed in [41], [42]), to regulate osteocalcin expression [37]. Runx2/Cbfa1 can in turn upregulate further C/EBPδ expression in osteoblasts [43]. The association of C/EBPβ and δ function with Runx2/Cbfa1 presents a critical linkage between these C/EBP factors and osteoblast differentiation given that Runx2/Cbfa1 is important for osteoblast responsiveness to extracellular matrix signals and is required for expression of genes associated with osteoblast differentiation [44]. Mice with a null mutation of runx2/cbfa1 have a complete absence of bone [45] and humans with cleidocranial dysplasia (CCD, marked by short stature and bone hypoplasia) have been demonstrated to have deletions within the human runx2/cbfa1 gene [46].

In addition to regulating growth and differentiation, C/EBPβ is most well known for its ability to be an effector in the induction of genes responsive to LPS, IL-1, or IL-6 stimulation (reviewed in Ref. [6]). Osteoblasts express C/EBP-responsive genes, including IL-1β, IL-6, IL-8, MCP-1, and TNFα  ([2], [47], [48], [49], [50], reviewed in Ref. [51]). In addition, these cytokines can, in a positive feedback loop, further induce C/EBPβ mRNA levels [2]. In osteoblasts, C/EBPβ has been shown to be induced by cytokines such as IL-1 [52] and is implicated in the regulation of IL-6 transcription [53]. IL-6 is particularly important because it plays a critical role in bone homeostasis and is produced by osteoblasts basally and at enhanced levels in response to known inducers. IL-6 is an important activator of osteoclast activity. Factors that modulate bone resorption such as parathyroid hormone (PTH), PTH-related peptide, androgens, estrogens, and thyroid hormones are also known to correspondingly influence the expression of IL-6. Knockout of IL-6 gene expression in transgenic mice prevented bone loss after oophorectomy [54], further suggesting an important role for IL-6 in inducing bone resorption.

The clear involvement of C/EBP family members in the differentiation and differentiated function of osteoblasts led us to further examine their activity in the temporal context of differentiating MC-3T3-E1 cells. In this report, we have found downregulation of C/EBP DNA-binding activity with mouse osteoblast differentiation. C/EBPβ was found to be the major component of C/EBP DNA-binding activity in proliferating MC3T3-E1 osteoblasts, although differentiating cells displayed greatly reduced C/EBP DNA-binding activity as well as reduced C/EBPβ protein levels. Furthermore, MC3T3-E1 cells stably expressing C/EBPβ LAP from a retroviral vector displayed enhanced proliferation and correspondingly suppressed differentiation as indicated by decreased alkaline phosphatase activity, a marker of osteoblast differentiation.