MLK3 regulates bone development downstream of the faciogenital dysplasia protein FGD1 in mice

Abstract

Mutations in human FYVE, RhoGEF, and PH domain-containing 1 (FGD1) cause faciogenital dysplasia (FGDY; also known as Aarskog syndrome), an X-linked disorder that affects multiple skeletal structures. FGD1 encodes a guanine nucleotide exchange factor (GEF) that specifically activates the Rho GTPase CDC42. However, the mechanisms by which mutations in FGD1 affect skeletal development are unknown. Here, we describe what we believe to be a novel signaling pathway in osteoblasts initiated by FGD1 that involves the MAP3K mixed-lineage kinase 3 (MLK3). We observed that MLK3 functions downstream of FGD1 to regulate ERK and p38 MAPK, which in turn phosphorylate and activate the master regulator of osteoblast differentiation, Runx2. Mutations in FGD1 found in individuals with FGDY ablated its ability to activate MLK3. Consistent with our description of this pathway and the phenotype of patients with FGD1 mutations, mice with a targeted deletion of Mlk3 displayed multiple skeletal defects, including dental abnormalities, deficient calvarial mineralization, and reduced bone mass. Furthermore, mice with knockin of a mutant Mlk3 allele that is resistant to activation by FGD1/CDC42 displayed similar skeletal defects, demonstrating that activation of MLK3 specifically by FGD1/CDC42 is important for skeletal mineralization. Thus, our results provide a putative biochemical mechanism for the skeletal defects in human FGDY and suggest that modulating MAPK signaling may benefit these patients. IntroductionIn 1970, Aarskog described an X-linked recessive syndrome characterized by an upturned nose, short stature, multiple dental defects, delayed skeletal age, and multiple bone malformations (1, 2). Later work confirmed these observations, naming the disorder faciogenital dysplasia (FGDY) or Aarskog-Scott syndrome and identified the gene mutated as FYVE, RhoGEF, and PH domain-containing 1 (FGD1) (3). FGD1 encodes a member of the guanine nucleotide exchange factor (GEF) family, which catalyses the exchange of GDP for GTP and promotes the activity of Rho family GTPases (3-6). More than 16 distinct FGD1 mutations have been reported to cosegregate with FGDY. These mutations include deletions and premature truncations, implying that a loss of FGD1 function underlies FGDY (6, 7). Despite these insights into the genetic basis of FGDY, it remains unclear how FGD1 affects bone development.Expression analysis demonstrates that FGD1 is highly expressed in osteoblasts, suggesting that FGD1 signaling plays a critical role in osteoblast differentiation and function (8). Microinjection studies show that FGD1 specifically activates CDC42, a member of the Rho (Ras homology) family of GTPase proteins (4, 5). A

The p38α MAPK positively regulates osteoblast function and postnatal bone acquisition

“…This hypothesis has been confirmed by in vitro analyses demonstrating that p38a regulates early osteoblast differentiation whereas p38b controls late osteoblast maturation [12]. Another MAP3K, mixed-lineage kinase 3 (MLK3) acting downstream of faciogenital dysplasia protein 1, has also been identified as a regulator of osteoblastogenesis and bone development by modulating ERK and p38 MAPK pathways [13]. In addition, the genetic disruption of the neighbor of Brca1 gene-encoding Nbr1, a receptor for selective autophagosomal degradation of ubiquitinated target proteins, has been shown to induce an age-dependent increase in bone mass due to continuous osteoblastic p38 MAPK activation and subsequent stimulated bone formation [14].…”

“…Furthermore, several signaling pathways have been shown to regulate postnatal osteoblast function including Ihh [26], Wnt/Lrp5 [27], TGFb-BMP/Smads [28][29][30], BMP/MEKK2/MEK5-7/Jnk [31], b-adrenergic [32], IGF-1 [16], insulin [33] and PTH signaling [34]. The p38 MAPK pathway has been recently shown to be an essential regulator of osteoblastogenesis during skeletogenesis [12,13]. Indeed, mice lacking MKK3 and MKK6 (upstream activators of p38 MAPK) exhibited low bone mass phenotype at 3 weeks of age, due to impaired Fig.…”

Focus on the p38 MAPK signaling pathway in bone development and maintenance

“…Pre-osteoblasts lacking TAK1 fail to differentiate into mature osteoblasts and have defective function. 34 Another MAP3K, MLK3, has been found to act downstream of faciogenital dysplasia protein 1 (FGD1) and has been identified as a physiologic regulator of osteoblastogenesis and bone development by modulating ERK and p38 MAPK activity 39 ( Figure 2). Mlk3 À / À mutant mice show dental abnormalities, defective mineralization of the calvarium and osteopenia, an equivalent skeletal phenotype in comparison with that of patients with mutations in FGD1.…”

“…This skeletal phenotype is associated with a significant decrease in bone formation and reduced expressions of osteoblast maker genes such as collagen 1 and osteocalcin. 39 In the absence of MLK3, the defective osteoblast differentiation is associated with impaired activation of ERK and p38 MAPK, and reduced RUNX2 phosphorylation. 39 Targets of p38 MAPK in osteoblasts.…”

“…39 In the absence of MLK3, the defective osteoblast differentiation is associated with impaired activation of ERK and p38 MAPK, and reduced RUNX2 phosphorylation. 39 Targets of p38 MAPK in osteoblasts. The similar cleidocranial dysplasia and osteopenia observed in mice lacking TAK1 in preosteoblasts and Runx2 þ / À mice have suggested that signaling pathways downstream of TAK1 may regulate RUNX2 activity.…”