Type VI Collagen Deficiency Causes Enhanced Periodontal Tissue Destruction.

The periodontal ligament (PDL) is a fibrillar connective tissue that lies between the alveolar bone and the tooth and is composed of highly specialized extracellular matrix (ECM) molecules and a heterogeneous population of cells that are responsible for collagen formation, immune response, bone formation, and chewing force sensation. Type VI collagen (COL6), a widely distributed ECM molecule, plays a critical role in the structural integrity and mechanical properties of various tissues including muscle, tendon, bone, cartilage, and skin. However, its role in the PDL remains largely unknown. Our study shows that deficiency of COL6 impairs PDL fibrillogenesis and exacerbates tissue destruction in ligature-induced periodontitis (LIP). We found that COL6-deficient mice exhibited increased bone loss and degraded PDL in LIP and that fibroblasts expressing high levels of Col6α2 are pivotal in ECM organization and cell-ECM interactions. Moreover, COL6 deficiency in the PDL led to an increased number of fibroblasts geared toward the inflammatory response. We also observed that cultured COL6-deficient fibroblasts from the PDL exhibited decreased expression of genes related to collagen fiber turnover and ECM organization as well as migration and proliferation. Our findings suggest that COL6 plays a crucial role in the PDL, influencing fibroblast function in fibrillogenesis and affecting the immune response in periodontitis. These insights advance our understanding of the molecular mechanisms underlying PDL maturation and periodontal disease.

WISP1/CCN4 aggravates cartilage degeneration in experimental osteoarthritis.

OBJECTIVE: Increased Wisp1 expression was previously reported in experimental and human osteoarthritis (OA). Moreover, adenoviral overexpression of Wisp1 in naïve mouse knee joints resulted in early OA-like cartilage lesions. Here, we determined how the matricellular protein WISP1 is involved in the pathology that occurs in the complex osteoarthritic environment with aging and experimental OA in wild type (WT) and Wisp1-/- mice. METHODS: WT and Wisp1-/- mice were aged or experimental OA was induced with intraarticular collagenase injection, destabilization of the medial meniscus (DMM) or anterior cruciate ligament transection (ACLT). Joint pathology was assessed using histology and microCT. Protease expression was evaluated with qRT-PCR and activity was determined by immunohistochemical staining of the aggrecan neoepitope NITEGE. Protease expression in human end-stage OA synovial tissue was determined with qRT-PCR after stimulation with WISP1. RESULTS: With aging, spontaneous cartilage degeneration in Wisp1-/- was not decreased compared to their WT controls. However, we observed significantly decreased cartilage degeneration in Wisp1-/- mice after induction of three independent experimental OA models. While the degree of osteophyte formation was comparable between WT and Wisp1-/- mice, increased cortical thickness and reduced trabecular spacing was observed in Wisp1-/- mice. In addition, we observed decreased MMP3/9 and ADAMTS4/5 expression in Wisp1-/- mice, which was accompanied by decreased levels of NITEGE. In line with this, stimulation of human OA synovium with WISP1 increased the expression of various proteases. CONCLUSIONS: WISP1 plays an aggravating role in the development of post-traumatic experimental OA.

Fibromodulin and Biglycan Modulate Periodontium through TGFß/BMP Signaling.

A full understanding of the key regulators controlling periodontal development and homeostasis is necessary for the design of improved periodontal regenerative therapies. Small leucine-rich proteoglycans (SLRPs) are extracellular matrix molecules suggested to regulate collagen organization and cell signaling. Mice with double-deficiency of 2 SLRPs, fibromodulin and biglycan (dKO), acquire skeletal abnormalities, but their roles in regulating the periodontium remain undefined and were the focus of our studies. Transmission electron microscopy studies showed abnormal collagen fibrils in the periodontal ligament (PDL) and altered remodeling of alveolar bone in dKO mice. Immunohistochemistry (IHC) revealed increased staining of SLRPs (asporin, lumican, and decorin) and dentin matrix protein-1 (DMP1, a mechanosensory/osteocyte marker), while osteoblast markers, bone sialoprotein and osteopontin, remained unchanged. Disruption of homeostasis was further evidenced by increased expression of receptor-activator of nuclear factor-κB ligand (RANKL) and elevated numbers of osteoclasts, especially noted around the alveolar bone of molars (buccal side) and incisors. Polymerase chain reaction (PCR) array revealed hyperactive transforming growth factors beta/bone morphogenetic protein (TGFß/BMP) signaling in dKO PDL tissues, which was further confirmed by elevated expression of phosphorylated Smad5 (p-Smad5) by IHC in dKO PDL. These studies highlight the importance of SLRPs in maintaining periodontal homeostasis through regulation of TGFß/BMP signaling, matrix turnover, and collagen organization.

The public health challenge of early growth failure in India.

Recent recognition of the early onset and high prevalence of wasting (30%) and stunting (20%) among infants 0-5 months in India draws attention to the need to understand the causes and develop prevention strategies. Such growth failure has dire consequences in the short (increased mortality) and long-term (loss of human capital and increased risk of chronic diseases). Food interventions before 6 months will increase morbidity/mortality through contamination in settings of poor sanitation and hygiene. Waiting to improve nutrition only after the initiation of complementary feeding at 6 months is a missed opportunity and may permanently alter life trajectory and potential. This underscores the importance of maternal nutrition. Iron and folic acid and protein energy supplementation during pregnancy are interventions that can improve maternal nutrition and birth outcomes. Maternal supplementation during lactation should be considered as a means to improve maternal and child outcomes, although the evidence needs strengthening. Support and counseling are also required to improve maternal diets and promote exclusive breastfeeding. Programs focused on improving maternal nutrition across the continuum of preconception, pregnancy and lactation are likely to have the greatest impact as mothers are central gatekeepers to the health and future of their children.

Impact of maternal and neonatal iron status on placental transferrin receptor expression in pregnant adolescents.

OBJECTIVE: To elucidate the role of maternal and neonatal iron status on placental transferrin receptor (TfR) expression. STUDY DESIGN AND OUTCOMES: Ninety-two healthy pregnant adolescents (ages 14-18 years) were followed across pregnancy. Maternal iron status (hemoglobin, hematocrit, serum ferritin, TfR, and total body iron) was assessed in mid-gestation (21-25 wks) and at delivery in the mother and neonate. Placental TfR protein expression was assessed by western blot in placental tissue collected at delivery. RESULTS: Placental TfR expression was inversely associated with maternal iron status at mid-gestation (hemoglobin p = 0.046, R(2) = 0.1 and hematocrit p = 0.005, R(2) = 0.24) and at delivery (serum ferritin p = 0.02, R(2) = 0.08 and total body iron p = 0.02, R(2) = 0.07). Mothers with depleted body iron stores had significantly greater placental expression of TfR than mothers with body iron stores greater than zero (p = 0.003). Neonatal iron stores were also inversely associated with the expression of placental TfR (p = 0.04, R(2) = 0.06). Neonates with serum ferritin values ≤ 34 µg/L had significantly greater protein expression of placental TfR compared to neonates with cord serum ferritin values >34 µg/L (p = 0.01). CONCLUSIONS: Expression of placental TfR is associated with both maternal and neonatal iron demands. Increased expression of placental TfR may be an important compensatory mechanism in response to iron deficiency in otherwise healthy pregnant women.

Phenotypic characterization of epiphycan-deficient and epiphycan/biglycan double-deficient mice.

OBJECTIVE: To characterize the in vivo role epiphycan (Epn) has in cartilage development and/or maintenance. METHODS: Epn-deficient mice were generated by disrupting the Epn gene in mouse embryonic stem cells. Epn/biglycan (Bgn) double-deficient mice were produced by crossing Epn-deficient mice with Bgn-deficient mice. Whole knee joint histological sections were stained using van Gieson or Fast green/Safranin-O to analyze collagen or proteoglycan content, respectively. Microarray analysis was performed to detect gene expression changes within knee joints. RESULTS: Epn-deficient and Epn/Bgn double-deficient mice appeared normal at birth. No significant difference in body weight or femur length was detected in any animal at 1 month of age. However, 9-month Epn/Bgn double-deficient mice were significantly lighter and had shorter femurs than wild type mice, regardless of gender. Male Epn-deficient mice also had significantly shorter femurs than wild type mice at 9 months. Most of the deficient animals developed osteoarthritis (OA) with age; the onset of OA was observed earliest in Epn/Bgn double-deficient mice. Message RNA isolated from Epn/Bgn double-deficient knee joints displayed increased matrix protein expression compared with wild type mice, including other small leucine-rich proteoglycan (SLRP) members such as asporin, fibromodulin and lumican. CONCLUSION: Similar to other previously studied SLRPs, EPN plays an important role in maintaining joint integrity. However, the severity of the OA phenotype in the Epn/Bgn double-deficient mouse suggests a synergy between these two proteins. These data are the first to show a genetic interaction involving class I and class III SLRPs in vivo.

Potential roles for the small leucine-rich proteoglycans biglycan and fibromodulin in ectopic ossification of tendon induced by exercise and in modulating rotarod performance.

We present a detailed comparison of ectopic ossification (EO) found in tendons of biglycan (Bgn), fibromodulin (Fmod) single and double Bgn/Fmod-deficient (DKO) mice with aging. At 3 months, Fmod KO, Bgn KO and DKO displayed torn cruciate ligaments and EO in their quadriceps tendon, menisci and cruciate and patellar ligaments. The phenotype was the least severe in the Fmod KO, intermediate in the Bgn KO and the most severe in the DKO. This condition progressed with age in all three mouse strains and resulted in the development of large supernumerary sesmoid bones. To determine the role of exercise in the extent of EO, we subjected normal and DKO mice to a treadmill exercise 3 days a week for 4 weeks. In contrast to previous findings using more rigorous exercise regimes, the EO in moderately exercised DKO was decreased compared with unexercised DKO mice. Finally, DKO and Bgn KO mice tested using a rotarod showed a reduced ability to maintain their grip on a rotating cylinder compared with wild-type controls. In summary, we show (1) a detailed description of EO formed by Bgn, Fmod or combined depletion, (2) the role of exercise in modulating EO and (3) that Bgn and Fmod are critical in controlling motor function.

Biglycan is required for adaptive remodeling after myocardial infarction.

BACKGROUND: After myocardial infarction (MI), extensive remodeling of extracellular matrix contributes to scar formation and preservation of hemodynamic function. On the other hand, adverse and excessive extracellular matrix remodeling leads to fibrosis and impaired function. The present study investigates the role of the small leucine-rich proteoglycan biglycan during cardiac extracellular matrix remodeling and cardiac hemodynamics after MI. METHODS AND RESULTS: Experimental MI was induced in wild-type (WT) and bgn(-/0) mice by permanent ligation of the left anterior descending coronary artery. Biglycan expression was strongly increased at 3, 7, and 14 days after MI in WT mice. bgn(-/0) mice showed increased mortality rates after MI as a result of frequent left ventricular (LV) ruptures. Furthermore, tensile strength of the LV derived from bgn(-/0) mice 21 days after MI was reduced as measured ex vivo. Collagen matrix organization was severely impaired in bgn(-/0) mice, as shown by birefringence analysis of Sirius red staining and electron microscopy of collagen fibrils. At 21 days after MI, LV hemodynamic parameters were assessed by pressure-volume measurements in vivo to obtain LV end-diastolic pressure, end-diastolic volume, and end-systolic volume. bgn(-/0) mice were characterized by aggravated LV dilation evidenced by increased LV end-diastolic volume (bgn(-/0), 111+/-4.2 microL versus WT, 96+/-4.4 microL; P<0.05) and LV end-diastolic pressure (bgn(-/0), 24+/-2.7 versus WT, 18+/-1.8 mm Hg; P<0.05) and severely impaired LV function (EF, bgn(-/0), 12+/-2% versus WT, 21+/-4%; P<0.05) 21 days after MI. CONCLUSIONS: Biglycan is required for stable collagen matrix formation of infarct scars and for preservation of cardiac hemodynamic function.

Application of local heat induces capillary recruitment in the Pallid bat wing.

Skin blood flow increases in response to local heat due to sensorineural and nitric oxide (NO)-mediated dilation. It has been previously demonstrated that arteriolar dilation is inhibited with NO synthase (NOS) blockade. Flow, nonetheless, increases with local heat. This implies that the previously unexamined nonarteriolar responses play a significant role in modulating flow. We thus hypothesized that local heating induces capillary recruitment. We heated a portion (3 cm2) of the Pallid bat wing from 25 degrees C to 37 degrees C for 20 min, and measured changes in terminal feed arteriole (approximately 25 microm) diameter and blood velocity to calculate blood flow (n = 8). Arteriolar dilation was reduced with NOS and sensorineural blockade using a 1% (wt/vol) NG-nitro-L-arginine methyl ester (L-NAME) and 2% (wt/vol) lidocaine solution (n = 8). We also measured changes in the number of perfused capillaries, and the time precapillary sphincters were open with (n = 8) and without (n = 8) NOS plus sensorineural blockade. With heat, the total number of perfused capillaries increased 92.7 +/- 17.9% (P = 0.011), and a similar increase occurred despite NOS plus sensorineural blockade 114.4 +/- 30.0% (P = 0.014). Blockade eliminated arteriolar dilation (-4.5 +/- 2.1%). With heat, the percent time precapillary sphincters remained open increased 32.3 +/- 6.0% (P = 0.0006), and this increase occurred despite NOS plus sensorineural blockade (34.1 +/- 5.8%, P = 0.0004). With heat, arteriolar blood flow increased (187.2 +/- 28.5%, P = 0.00003), which was significantly attenuated with NOS plus sensorineural blockade (88.6 +/- 37.2%, P = 0.04). Thus, capillary recruitment is a fundamental microvascular response to local heat, independent of arteriolar dilation and the well-documented sensorineural and NOS mechanisms mediating the response to local heat.

Small leucine-rich proteoglycans in the aging skeleton.

Small Leucine-Rich Proteoglyans (SLRPs) are major skeletal extracellular matrix (ECM) components that comprise a family of 13 members containing repeats of a leucine-rich motif. To examine SLRP function, we generated mice deficient in one or more member and analyzed them at the tissue, cell and molecular levels. This review outlines the novel research findings uncovered using these new animal models.

Local heat produces a shear-mediated biphasic response in the thermoregulatory microcirculation of the Pallid bat wing.

Investigators report that local heat causes an increase in skin blood flow consisting of two phases. The first is solely sensory neural, and the second is nitric oxide mediated. We hypothesize that mechanisms behind these two phases are causally linked by shear stress. Because microvascular blood flow, endothelial shear stress, and vessel diameters cannot be measured in humans, bat wing arterioles (26.6 +/- 0.3, 42.0 +/- 0.4, and 58.7 +/- 2.2 microm) were visualized noninvasively on a transparent heat plate via intravital microscopy. Increasing plate temperature from 25 to 37 degrees C increased flow in all three arterial sizes (137.1 +/- 0.3, 251.9 +/- 0.5, and 184.3 +/- 0.6%) in a biphasic manner. With heat, diameter increased in large arterioles (n = 6) by 8.7 +/- 0.03% within 6 min, medium arterioles (n = 8) by 19.7 +/- 0.5% within 4 min, and small arterioles (n = 8) by 31.6 +/- 2.2% in the first minute. Lidocaine (0.2 ml, 2% wt/vol) and NG-nitro-L-arginine methyl ester (0.2 ml, 1% wt/vol) were applied topically to arterioles (approximately 40 microm) to block sensory nerves, modulate shear stress, and block nitric oxide generation. Local heat caused only a 10.4 +/- 5.5% increase in diameter with neural blockade (n = 8) and only a 7.5 +/- 4.1% increase in diameter when flow was reduced (n = 8), both significantly lower than control (P < 0.001). Diameter and flow increases were significantly reduced with NG-nitro-L-arginine methyl ester application (P < 0.05). Our novel thermoregulatory animal model illustrates 1) regulation of shear stress, 2) a nonneural component of the first phase, and 3) a shear-mediated second phase. The time course of dilation suggests that early dilation of small arterioles increases flow and enhances second-phase dilation of the large arterioles.

Targeted disruption of two small leucine-rich proteoglycans, biglycan and decorin, excerpts divergent effects on enamel and dentin formation.

Small leucine-rich proteoglycans have been suggested to affect mineralization of dental hard tissues. To determine the functions of two of these small proteoglycans during the early stages of tooth formation, we characterized the dental phenotypes of biglycan (BGN KO) and decorin deficient (DCN KO) mice and compared them to that of wild type mice. Each targeted gene disruption resulted in specific effects on dentin and enamel formation. Dentin was hypomineralized in both knock out mice, although the effect was more prominent in the absence of decorin. Enamel formation was dramatically increased in newborn biglycan knockout mice but delayed in absence of decorin. Increased enamel formation in the former case resulted from an upregulation of amelogenin synthesis whereas delayed enamel formation in the later case was most probably an indirect consequence of the high porosity of the underlying dentin. Enamelin expression was unchanged in BGN KO, and reduced in DCN KO. Dentin sialoprotein (DSP), a member of the family of phosphorylated extracellular matrix proteins that play a role in dentinogenesis, was overexpressed in BGN-KO odontoblasts and in the sub-odontoblastic layer. In contrast, a decreased expression of DSP was detected in DCN KO. Dentin matrix protein-1 (DMP-1), bone sialoprotein (BSP) and osteopontin (OPN) were upregulated in BGN KO and downregulated in the DCN KO. Despite the strong effects induced by these deficiencies in newborn mice, no significant difference was detected between the three genotypes in adult mice, suggesting that the effects reported here in newborn mice are transient and subjected to self-repair.

Accelerated osteoarthritis in the temporomandibular joint of biglycan/fibromodulin double-deficient mice.

OBJECTIVE: To investigate whether the absence of biglycan and fibromodulin, two proteoglycans expressed in cartilage, bone and tendon, resulted in accelerated osteoarthritis in the temporomandibular joint (TMJ). METHODS: Histological sections of TMJ from 3-, 6-, 9- and 18-month-old wild-type (WT) and biglycan/fibromodulin double-deficient (DKO) mice were compared. Immuno-stainings for biglycan, fibromodulin and proliferating cell nuclear antigen (PCNA) were performed. RESULTS: Biglycan and fibromodulin were highly expressed in the disc and articular cartilage of the TMJ. At 3 months of age, both WT and DKO presented early signs of cartilage degeneration visible as small acellular areas under the articular surfaces and superficial waving. From 6 months of age, DKOs developed accelerated osteoarthritis compared to WT. At 6 months, small vertical clefts in the condylar cartilage and partial disruption of the disk were visible in the DKO. In addition, chondrocytes had lost their regular columnar organization to form clusters. At 9 months, these differences were even more pronounced. At 18 months, extended cartilage erosion was visible in DKOs when by comparison the thickness of the articular cartilage in WT controls was basically intact. PCNA staining was stronger in 3-month-old WT TMJ fibrocartilage than in 3-month-old DKO TMJ fibrocartilage suggesting that chondrocyte proliferation might be impaired in DKOs. CONCLUSION: The biglycan/fibromodulin double knock-out mouse constitutes a useful animal model to decipher the pathobiology of osteoarthritis in the TMJ.

Constitutively active PTH/PTHrP receptor in odontoblasts alters odontoblast and ameloblast function and maturation.

Parathyroid hormone (PTH)-related protein (PTH-rP) is an important autocrine/paracrine attenuator of programmed cell differentiation whose expression is restricted to the epithelial layer in tooth development. The PTH/PTHrP receptor (PPR) mRNA in contrast is detected in the dental papilla, suggesting that PTHrP and the PPR may modulate epithelial-mesenchymal interactions. To explore the possible interactions, we studied the previously described transgenic mice in which a constitutively active PPR is targeted to osteoblastic cells. These transgenic mice have a vivid postnatal bone and tooth phenotype, with normal tooth eruption but abnormal, widened crowns. Transgene mRNA expression was first detected at birth in the dental papilla and, at 1 week postnatally, in odontoblasts. There was no transgene expression in ameloblasts or in other epithelial structures. Prenatally, transgenic molars and incisors revealed no remarkable change. By the age of 1 week, the dental papilla was widened, with disorganization of the odontoblastic layer and decreased dentin matrix. In addition, the number of cusps was abnormally increased, the ameloblastic layer disorganized, and enamel matrix decreased. Odontoblastic and, surprisingly, ameloblastic cytodifferentiation was impaired, as shown by in situ hybridization and electron microscopy. Interestingly, ameloblastic expression of Sonic Hedgehog, a major determinant of ameloblastic cytodifferentiation, was dramatically altered in the transgenic molars. These data suggest that odontoblastic activation of the PPR may play an important role in terminal odontoblastic and, indirectly, ameloblastic cytodifferentiation, and describe a useful model to study how this novel action of the PPR may modulate mesenchymal/epithelial interactions at later stages of tooth morphogenesis and development.

Phenotypic effects of biglycan deficiency are linked to collagen fibril abnormalities, are synergized by decorin deficiency, and mimic Ehlers-Danlos-like changes in bone and other connective tissues.

Decorin (dcn) and biglycan (bgn), two members of the family of small leucine-rich proteoglycans (SLRPs), are the predominant proteoglycans expressed in skin and bone, respectively. Targeted disruption of the dcn gene results in skin laxity and fragility, whereas disruption of the bgn gene results in reduced skeletal growth and bone mass leading to generalized osteopenia, particularly in older animals. Here, we report that bgn deficiency leads to structural abnormality in collagen fibrils in bone, dermis, and tendon, and to a "subclinical" cutaneous phenotype with thinning of the dermis but without overt skin fragility. A comparative ultrastructural study of different tissues from bgn- and dcn-deficient mice revealed that bgn and dcn deficiency have similar effects on collagen fibril structure in the dermis but not in bone. Ultrastructural and phenotypic analysis of newly generated bgn/dcn double-knockout (KO) mice revealed that the effects of dcn and bgn deficiency are additive in the dermis and synergistic in bone. Severe skin fragility and marked osteopenia characterize the phenotype of double-KO animals in which progeroid changes are observed also in the skin. Ultrastructural analysis of bone collagen fibrils in bone of double-KO mice reveals a complete loss of the basic fibril geometry with the emergence of marked "serrated fibril" morphology. The phenotype of the double-KO animal mimics directly the rare progeroid variant of human Ehlers-Danlos syndrome (EDS), in which skin fragility, progeroid changes in the skin (reduced hypodermis), and osteopenia concur as a result of impaired glycosaminoglycan (GAG) linking to bgn and dcn core proteins. Our data show that changes in collagen fibril morphology reminiscent of those occurring in the varied spectrum of human EDS are induced by both bgn deficiency and den deficiency in mice. The effects of an individual SLRP deficiency are tissue specific, and the expression of a gross phenotype depends on multiple variables including level of expression of individual SLRPs in different tissues and synergisms between different SLRPs (and likely other macromolecules) in determining matrix structure and functional properties.

The human tuftelin gene: cloning and characterization.

Tuftelin has been suggested to play an important role during the development and mineralization of enamel. We isolated the full-length human tuftelin cDNA using reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (5' RACE and 3' RACE) methods. Sequence analysis of the tuftelin cDNA revealed an open reading frame of 1170 bp encoding a 390 amino acid protein with a molecular mass of 44.3 kDa and an isoelectric point of 5.7. The human tuftelin protein shares 89 and 88% amino acid sequence identity with the bovine and mouse tuftelin, respectively. It contains a coiled-coil region, recently reported to be involved with tuftelin self-assembly and with the interaction of tuftelin with TIP39 (a novel tuftelin interacting protein). Detailed DNA analysis of the cloned genomic DNA revealed that the human tuftelin gene contains 13 exons and is larger than 26 kb. Two alternatively spliced tuftelin mRNA transcripts have now been identified in the human tooth bud, one lacking exon 2, and the other lacking exon 2 and exon 3. Primer extension analysis, corroborated by RT-PCR and DNA sequencing, revealed multiple transcription initiation sites. The cloned 1.6 kb promoter region contained several GC boxes and several transcription factor binding sites such as those for activator protein 1 and stimulatory protein 1. Our blast search of the human and mouse expressed sequence tag data bases, as well as our RT-PCR and DNA sequencing results, and a previous study using Northern blot analysis revealed that tuftelin cDNA sequences are also expressed in normal and cancerous non-mineralizing soft tissues, suggesting that tuftelin has a universal function. We have now identified and characterized different alternatively spliced mouse tuftelin mRNAs in several non-mineralizing tissues. These results provide an important baseline for future understanding of the biological role of tuftelin.

The urokinase plasminogen activator receptor-associated protein/endo180 is coexpressed with its interaction partners urokinase plasminogen activator receptor and matrix metalloprotease-13 during osteogenesis.

The urokinase plasminogen activator receptor-associated protein/Endo180 (uPARAP/Endo180) is a newly discovered member of the macrophage mannose receptor family that was reported to interact with ligand-bound urokinase plasminogen activator receptor (uPAR), matrix metalloprotease-13 (MMP-13), and collagen V on the cell surface. We have determined the sites of expression of this novel receptor during murine postimplantation development. uPARAP/Endo180 was expressed in all tissues undergoing primary ossification, including the developing bones of the viscerocranium and calvarium that ossify intramembranously, and developing long bones undergoing endochondral ossification. uPARAP/Endo180 mRNA was expressed by both immature osteoblasts and by mature osteocalcin-producing osteoblasts-osteocytes, and was coexpressed with MMP-13. Interestingly, osteoblasts also expressed uPAR. Besides bone-forming tissues, uPARAP/Endo180 expression was detected only in a mesenchymal condensation of the midbrain and in the developing lungs. The data suggest a function of this novel protease receptor in bone development, possibly mediated through its interactions with uPAR, MMP-13, or collagen V.

Flexible structures of SIBLING proteins, bone sialoprotein, and osteopontin.

Bone sialoprotein (BSP) and osteopontin (OPN) are two members of the SIBLING (Small Integrin-Binding LIgand, N-linked Glycoprotein) family of genetically related proteins that are clustered on human chromosome 4. We present evidence that this entire family is the result of duplication and subsequent divergent evolution of a single ancient gene. The solution structures of these two post-translationally modified recombinant proteins were solved by one dimensional proton NMR and transverse relaxation times. The polypeptide backbones of both free BSP and OPN rapidly sample an ensemble of conformations consistent with them both being completely unstructured in solution. This flexibility appears to enable these relatively small glycoproteins to rapidly associate with a number of different binding partners including other proteins as well as the mineral phase of bones and teeth. These proteins often function by bridging two proteins of fixed structures into a biologically active complex.

Double FYVE-containing protein 1 (DFCP1): isolation, cloning and characterization of a novel FYVE finger protein from a human bone marrow cDNA library.

Double FYVE-containing protein 1 (DFCP1) encodes a 777 amino acid protein that contains: (1) an N-terminal Cys-His cluster with some homology to many zinc finger domains; (2) a consensus sequence consistent with an ATP/GTP binding site; and (3) a C-terminal domain unique because it contains two zinc-binding FYVE domains. The gene, ZNFN2A1 (GenBank accession no. AF251025) was localized to chromosome 14q22-q24 and shown to be composed of 11 exons. Northern blot analysis revealed the presence of three different mRNA transcripts (4.2, 3 and 1.2kb). The two longer transcripts appear to be expressed in a variety of different tissues, especially in endocrine tissues, while the shorter messenger is limited to testis. Both of the larger transcripts are unusual due to the presence of a 463bp long 5' UTR. Furthermore, the 4.2kb transcript contains a non-standard polyadenylation consensus sequence while the 3kb transcript contains a standard consensus sequence but within the open reading frame. Following in vitro transfection of a DFCP1-containing expression construct, confocal microscopy studies showed a vesicular distribution of DFCP1 suggesting that this protein, like other FYVE-containing proteins, might be involved in membrane trafficking.