Pelvic organ prolapse (POP) is a common condition affecting almost half of women over the age of 50. The molecular and cellular mechanisms underlying this condition, however, remain poorly understood. Here we have reported that fibulin-5, an integrin-binding matricellular protein that is essential for elastic fiber assembly, regulated the activity of MMP-9 to maintain integrity of the vaginal wall and prevented development of POP. In murine vaginal stromal cells, fibulin-5 inhibited the β 1 integrin-dependent, fibronectin-mediated upregulation of MMP-9. Mice in which the integrin-binding motif was mutated to an integrin-disrupting motif (Fbln5 RGE/RGE ) exhibited upregulation of MMP-9 in vaginal tissues. In contrast to fibulin-5 knockouts (Fbln5 -/-), Fbln5 RGE/RGE mice were able to form intact elastic fibers and did not exhibit POP. However, treatment of mice with β-aminopropionitrile (BAPN), an inhibitor of matrix cross-linking enzymes, induced subclinical POP. Conversely, deletion of Mmp9 in Fbln5 -/-mice significantly attenuated POP by increasing elastic fiber density and improving collagen fibrils. Vaginal tissue samples from pre-and postmenopausal women with POP also displayed significantly increased levels of MMP-9. These results suggest that POP is an acquired disorder of extracellular matrix and that therapies targeting matrix proteases may be successful for preventing or ameliorating POP in women. IntroductionPelvic organ prolapse (POP) is characterized by abnormal protrusion of female pelvic organs involving the uterus, bladder, and vagina (reviewed in refs. 1-3). Epidemiologic studies indicate that (a) vaginal birth, aging, and increased body mass index are major risk factors for the development of POP and (b) more than 1 pathology may be involved to exhibit full anatomical loss of support. Although approximately 11% of women have surgery for POP or urinary incontinence in their lifetimes (4), to date, effective therapies to prevent progression of POP have not been established, thereby imposing profound social and financial burden to affected individuals (3).Despite a difference in anatomical position of pelvic organs relative to the body axis and pelvic floor, rodent models of POP have provided important tools to study underlying mechanisms of prolapse. In contrast to an observation that rectal prolapse is frequently associated with the presence of chronic inflammatory bowel disease (5), POP has been found in animals with defective ECM proteins, including fibulin-3, fibulin-5, and lysyl oxidaselike-1 (LOXL-1) (an enzyme that predominantly catalyzes crosslinking of elastin) (6-8). Interestingly, these proteins are abundantly expressed in the vaginal wall and involved in synthesis and assembly of elastic fibers. It is also known that uterosacral ligaments support the vaginal wall and that Hoxa11 is essential for formation of uterosacral ligaments in mice (9). Hoxa11-deficient mice exhibit increased mobility of the uterus; however,

Rationale: Loss of fibulin-4 during embryogenesis results in perinatal lethality because of aneurysm rupture, and defective elastic fiber assembly has been proposed as an underlying cause for the aneurysm phenotype. However, aneurysms are never seen in mice deficient for elastin, or for fibulin-5, which absence also leads to compromised elastic fibers. Objective: We sought to determine the mechanism of aneurysm development in the absence of fibulin-4 and establish the role of fibulin-4 in aortic development. Methods and Results: We generated germline and smooth muscle cell (

Hand proteins are evolutionally conserved basic helix-loop-helix (bHLH) transcription factors implicated in development of neural crest-derived tissues, heart and limb. Hand1 is expressed in the distal (ventral) zone of the branchial arches, whereas the Hand2 expression domain extends ventrolaterally to occupy two-thirds of the mandibular arch. To circumvent the early embryonic lethality of Hand1 or Hand2-null embryos and to examine their roles in neural crest development, we generated mice with neural crest-specific deletion of Hand1 and various combinations of mutant alleles of Hand2. Ablation of Hand1 alone in neural crest cells did not affect embryonic development, however, further removing one Hand2 allele or deleting the ventrolateral branchial arch expression of Hand2 led to a novel phenotype presumably due to impaired growth of the distal midline mesenchyme. Although we failed to detect changes in proliferation or apoptosis between the distal mandibular arch of wild-type and Hand1/Hand2 compound mutants at embryonic day (E)10.5, dysregulation of Pax9, Msx2 and Prx2 was observed in the distal mesenchyme at E12.5. In addition, the inter-dental mesenchyme and distal symphysis of Meckel's cartilage became hypoplastic, resulting in the formation of a single fused lower incisor within the hypoplastic fused mandible. These findings demonstrate the importance of Hand transcription factors in the transcriptional circuitry of craniofacial and tooth development.

Members of the basic helix-loop-helix (bHLH) family of transcription factors regulate the specification and differentiation of numerous cell types during embryonic development. Hand1 and Hand2 are expressed by a subset of neural crest cells in the anterior branchial arches and are involved in craniofacial development. However, the precise mechanisms by which Hand proteins mediate biological actions and regulate downstream target genes in branchial arches is largely unknown. Here, we report that Hand2 negatively regulates intramembranous ossification of the mandible by directly inhibiting the transcription factor Runx2, a master regulator of osteoblast differentiation. Hand proteins physically interact with Runx2, suppressing its DNA binding and transcriptional activity. This interaction is mediated by the N-terminal domain of the Hand protein and requires neither dimerization with other bHLH proteins nor DNA binding. We observed partial colocalization of Hand2 and Runx2 in the mandibular primordium of the branchial arch, and downregulation of Hand2 precedes Runx2-driven osteoblast differentiation. Hand2 hypomorphic mutant mice display insufficient mineralization and ectopic bone formation in the mandible due to accelerated osteoblast differentiation, which is associated with the upregulation and ectopic expression of Runx2 in the mandibular arch. Here, we show that Hand2 acts as a novel inhibitor of the Runx2-DNA interaction and thereby regulates osteoblast differentiation in branchial arch development.

Objective-Recent findings on the role of fibulin-5 (Fbln5) have provided substantial progress in understanding the molecular mechanism of elastic fiber assembly in vitro. However, little is known about differential roles of fibulins in the elastogenesis of blood vessels. Here, we generated double knockout mice for Fbln5 and Fbln2 (termed DKO) and examined the role of fibulins-2 and -5 in development and injury response of the blood vessel wall. Methods and Results-Fibulin-2 is distinctly located in the subendothelial matrix, whereas fibulin-5 is observed throughout the vessel wall. All of the elastic laminae, including the internal elastic lamina (IEL), were severely disorganized in DKO mice, which was not observed in single knockout mice for Fbln2 or Fbln5. Furthermore, DKO vessels displayed upregulation of vascular adhesion molecules, tissue factor expression, and thrombus formation with marked dilation and thinning of the vessel wall after carotid artery ligation-injury. Conclusions-Fibulin-2 and fibulin-5 cooperatively function to form the IEL during postnatal development by directing the assembly of elastic fibers, and are responsible for maintenance of the adult vessel wall after injury. The DKO mouse will serve as a unique animal model to test the effect of vessel integrity during various pathological insults. Key Words: internal elastic lamina Ⅲ vascular remodeling Ⅲ development Ⅲ injury T he internal elastic lamina (IEL) is located beneath the endothelium of blood vessels and forms the innermost elastic lamina. The IEL provides elasticity and recoil to the vessel wall, as well as functions as a physical barrier against chemical/mechanical stresses, preventing direct contact of plasma components to smooth muscle cells (SMCs). Several pathological processes have been shown to bring about disruption of the IEL. For example, increased mechanical forces and shear stress associated with angioplasty, enzymatic activation of matrix degrading enzymes in atherosclerosis, and abdominal aneurysms are all associated with disruption of the IEL and progression of vascular disease. [1][2][3] Molecular mechanisms of elastic fiber assembly have begun to be explored by the identification of elastic fiber-associated proteins and their biochemical interactions with elastin or the microfibrillar scaffold. 4 Members of the fibulin family of extracellular matrix (ECM) proteins, particularly fibulin-4 and fibulin-5, play essential roles in elastic fiber development. [5][6][7] We demonstrated that fibulin-5 preferentially binds the monomeric form of elastin, but not polymerized elastin. 8 Others have shown that fibulin-5 accelerates the self-aggregation process of elastin, called coacervation, 9 and fibulin-5 limits maturation of the coacervated elastin. 10 Among 5 of the known fibulins tested, fibulin-2 and fibulin-5 exhibit the highest binding affinity to elastin. 11 Biochemical interaction assays showed that fibulin-2 also binds numerous basement membrane (BM) proteins including nidogen, laminin, and fibronectin. 12 Recently,...

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The basic helix-loop-helix (bHLH) transcription factor Hand2 is required for growth and development of the heart, branchial arches and limb buds. To determine whether DNA binding is required for Hand2 to regulate the growth and development of these different embryonic tissues, we generated mutant mice in which the Hand2 locus was modified by a mutation (referred to as Hand2 EDE ) that abolished the DNA-binding activity of Hand2, leaving the remainder of the protein intact. In contrast to Hand2 null embryos, which display right ventricular hypoplasia and vascular abnormalities, causing severe growth retardation by E9.5 and death by E10.5, early development of the heart appeared remarkably normal in homozygous Hand2 EDE mutant embryos. These mutant embryos also lacked the early defects in growth of the branchial arches seen in Hand2 null embryos and survived up to 2 to 3 days longer than did Hand2 null embryos. However, Hand2 EDE mutant embryos exhibited growth defects in the limb buds similar to those of Hand2 null embryos. These findings suggest that Hand2 regulates tissue growth and development in vivo through DNA binding-dependent and -independent mechanisms.KEY WORDS: bHLH, Hand2, Heart development, Limb development, Craniofacial development Development 136, 933-942 (2009)

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