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Item A crystallin mutant cataract with mineral deposits(Elsevier, 2023) Minogue, Peter J.; Gao, Junyuan; Mathias, Richard T.; Williams, James C., Jr.; Bledsoe, Sharon B.; Sommer, Andre J.; Beyer, Eric C.; Berthoud, Viviana M.; Anatomy, Cell Biology and Physiology, School of MedicineConnexin mutant mice develop cataracts containing calcium precipitates. To test whether pathologic mineralization is a general mechanism contributing to the disease, we characterized the lenses from a nonconnexin mutant mouse cataract model. By cosegregation of the phenotype with a satellite marker and genomic sequencing, we identified the mutant as a 5-bp duplication in the γC-crystallin gene (Crygcdup). Homozygous mice developed severe cataracts early, and heterozygous animals developed small cataracts later in life. Immunoblotting studies showed that the mutant lenses contained decreased levels of crystallins, connexin46, and connexin50 but increased levels of resident proteins of the nucleus, endoplasmic reticulum, and mitochondria. The reductions in fiber cell connexins were associated with a scarcity of gap junction punctae as detected by immunofluorescence and significant reductions in gap junction-mediated coupling between fiber cells in Crygcdup lenses. Particles that stained with the calcium deposit dye, Alizarin red, were abundant in the insoluble fraction from homozygous lenses but nearly absent in wild-type and heterozygous lens preparations. Whole-mount homozygous lenses were stained with Alizarin red in the cataract region. Mineralized material with a regional distribution similar to the cataract was detected in homozygous lenses (but not wild-type lenses) by micro-computed tomography. Attenuated total internal reflection Fourier-transform infrared microspectroscopy identified the mineral as apatite. These results are consistent with previous findings that loss of lens fiber cell gap junctional coupling leads to the formation of calcium precipitates. They also support the hypothesis that pathologic mineralization contributes to the formation of cataracts of different etiologies.Item Connexin Mutants Cause Cataracts Through Deposition of Apatite(Frontiers Media, 2022-07-22) Minogue, Peter J.; Sommer, Andre J.; Williams, James C., Jr.; Bledsoe, Sharon B.; Beyer, Eric C.; Berthoud, Viviana M.; Anatomy, Cell Biology and Physiology, School of MedicineCataracts are lens opacities that are among the most common causes of blindness. It is commonly believed that cataracts develop through the accumulation of damage to lens proteins. However, recent evidence suggests that cataracts can result from calcium ion accumulation and the precipitation of calcium-containing salts. To test for the presence of precipitates and to identify their components, we studied the lenses of mice that develop cataracts due to mutations of connexin46 and connexin50. Micro-computed tomography showed the presence of radio-dense mineral in the mutant lenses, but not in wild-type lenses. Three-dimensional reconstructions of the scans showed that the distribution of the radio-dense mineral closely paralleled the location and morphology of the cataracts. The mutant lens homogenates also contained insoluble particles that stained with Alizarin red (a dye that stains Ca2+ deposits). Using attenuated total internal reflection micro–Fourier transform infrared spectroscopy, we identified the mineral as calcium phosphate in the form of apatite. Taken together, these data support the novel paradigm that cataracts are formed through pathological mineralization within the lens.Item Extract of Artemisia dracunculus L. Modulates Osteoblast Proliferation and Mineralization(MDPI, 2023-08-30) Scott, Matthew C.; Bourgeois, Aleah; Yu, Yongmei; Burk, David H.; Smith, Brenda J.; Floyd, Z. Elizabeth; Obstetrics and Gynecology, School of MedicineThiazolidinediones (TZD) significantly improve insulin sensitivity via action on adipocytes. Unfortunately, TZDs also degrade bone by inhibiting osteoblasts. An extract of Artemisia dracunculus L., termed PMI5011, improves blood glucose and insulin sensitivity via skeletal muscle, rather than fat, and may therefore spare bone. Here, we examine the effects of PMI5011 and an identified active compound within PMI5011 (2′,4′-dihydroxy-4-methoxydihydrochalcone, DMC-2) on pre-osteoblasts. We hypothesized that PMI5011 and DMC-2 will not inhibit osteogenesis. To test our hypothesis, MC3T3-E1 cells were induced in osteogenic media with and without PMI5011 or DMC-2. Cell lysates were probed for osteogenic gene expression and protein content and were stained for osteogenic endpoints. Neither compound had an effect on early stain outcomes for alkaline phosphatase or collagen. Contrary to our hypothesis, PMI5011 at 30 µg/mL significantly increases osteogenic gene expression as early as day 1. Further, osteogenic proteins and cell culture mineralization trend higher for PMI5011-treated wells. Treatment with DMC-2 at 1 µg/mL similarly increased osteogenic gene expression and significantly increased mineralization, although protein content did not trend higher. Our data suggest that PMI5011 and DMC-2 have the potential to promote bone health via improved osteoblast maturation and activity.Item Pyk2 and Megakaryocytes Regulate Osteoblast Differentiation and Migration Via Distinct and Overlapping Mechanisms(Wiley, 2016-06) Eleniste, Pierre P.; Patel, Vruti; Posritong, Sumana; Zero, Odette; Largura, Heather; Cheng, Ying-Hua; Himes, Evan R.; Hamilton, Matthew; Baughman, Jenna; Kacena, Melissa A.; Bruzzaniti, Angela; Department of Biomedical and Applied Sciences, School of DentistryOsteoblast differentiation and migration are necessary for bone formation during bone remodeling. Mice lacking the proline-rich tyrosine kinase Pyk2 (Pyk2-KO) have increased bone mass, in part due to increased osteoblast proliferation. Megakaryocytes (MKs), the platelet-producing cells, also promote osteoblast proliferation in vitro and bone-formation in vivo via a pathway that involves Pyk2. In the current study, we examined the mechanism of action of Pyk2, and the role of MKs, on osteoblast differentiation and migration. We found that Pyk2-KO osteoblasts express elevated alkaline phosphatase (ALP), type I collagen and osteocalcin mRNA levels as well as increased ALP activity, and mineralization, confirming that Pyk2 negatively regulates osteoblast function. Since Pyk2 Y402 phosphorylation is important for its catalytic activity and for its protein-scaffolding functions, we expressed the phosphorylation-mutant (Pyk2(Y402F) ) and kinase-mutant (Pyk2(K457A) ) in Pyk2-KO osteoblasts. Both Pyk2(Y402F) and Pyk2(K457A) reduced ALP activity, whereas only kinase-inactive Pyk2(K457A) inhibited Pyk2-KO osteoblast migration. Consistent with a role for Pyk2 on ALP activity, co-culture of MKs with osteoblasts led to a decrease in the level of phosphorylated Pyk2 (pY402) as well as a decrease in ALP activity. Although, Pyk2-KO osteoblasts exhibited increased migration compared to wild-type osteoblasts, Pyk2 expression was not required necessary for the ability of MKs to stimulate osteoblast migration. Together, these data suggest that osteoblast differentiation and migration are inversely regulated by MKs via distinct Pyk2-dependent and independent signaling pathways. Novel drugs that distinguish between the kinase-dependent or protein-scaffolding functions of Pyk2 may provide therapeutic specificity for the control of bone-related diseases.Item Pyk2 deficiency potentiates osteoblast differentiation and mineralizing activity in response to estrogen or raloxifene(Elsevier, 2018-10-15) Posritong, Sumana; Hong, Jung Min; Eleniste, Pierre P.; McIntyre, Patrick W.; Wu, Jennifer L.; Himes, Evan R.; Patel, Vruti; Kacena, Melissa A.; Bruzzaniti, Angela; Biomedical Sciences and Comprehensive Care, School of DentistryBone remodeling is controlled by the actions of bone-degrading osteoclasts and bone-forming osteoblasts (OBs). Aging and loss of estrogen after menopause affects bone mass and quality. Estrogen therapy, including selective estrogen receptor modulators (SERMs), can prevent bone loss and increase bone mineral density in post-menopausal women. Although investigations of the effects of estrogen on osteoclast activity are well advanced, the mechanism of action of estrogen on OBs is still unclear. The proline-rich tyrosine kinase 2 (Pyk2) is important for bone formation and female mice lacking Pyk2 (Pyk2-KO) exhibit elevated bone mass, increased bone formation rate and reduced osteoclast activity. Therefore, in the current study, we examined the role of estrogen signaling on the mechanism of action of Pyk2 in OBs. As expected, Pyk2-KO OBs showed significantly higher proliferation, matrix formation, and mineralization than WT OBs. In addition we found that Pyk2-KO OBs cultured in the presence of either 17β-estradiol (E2) or raloxifene, a SERM used for the treatment of post-menopausal osteoporosis, showed a further robust increase in alkaline phosphatase (ALP) activity and mineralization. We examined the possible mechanism of action and found that Pyk2 deletion promotes the proteasome-mediated degradation of estrogen receptor α (ERα), but not estrogen receptor β (ERβ). As a consequence, E2 signaling via ERβ was enhanced in Pyk2-KO OBs. In addition, we found that Pyk2 deletion and E2 stimulation had an additive effect on ERK phosphorylation, which is known to stimulate cell differentiation and survival. Our findings suggest that in the absence of Pyk2, estrogen exerts an osteogenic effect on OBs through altered ERα and ERβ signaling. Thus, targeting Pyk2, in combination with estrogen or raloxifene, may be a novel strategy for the prevention and/or treatment of bone loss diseases.