Osteoactivin promotes breast cancer metastasis to bone

The skeleton is a preferred site of metastasis in patients with disseminated breast cancer. We have used 4T1 mouse mammary carcinoma cells, which metastasize to bone from the mammary fat pads of immunocompetent mice, to identify novel genes involved in this process. In vivo selection of parental cells resulted in the isolation of independent, aggressively bone metastatic breast cancer populations with reduced metastasis to the lung. Gene expression profiling identified osteoactivin as a candidate that is highly and selectively expressed in aggressively bone metastatic breast cancer cells. These cells displayed enhanced migratory and invasive characteristics in vitro, the latter requiring sustained osteoactivin expression. Osteoactivin depletion in these cells, by small interfering RNA, also lead to a loss of matrix metalloproteinase-3 expression, whereas forced osteoactivin expression in parental 4T1 cells was sufficient to elevate matrix metalloproteinase-3 levels, suggesting that this matrix metalloproteinase may be an important mediator of osteoactivin function. Overexpression of osteoactivin in an independent, weakly bone metastatic breast cancer cell model significantly enhanced the formation of osteolytic bone metastases in vivo. Finally, high levels of osteoactivin expression in primary human breast cancers correlate with estrogen receptor-negative status and increasing tumor grade. Thus, we have identified osteoactivin as a protein that is expressed in aggressive human breast cancers and is capable of promoting breast cancer metastasis to bone.     

Escherichia coli HdeB is an acid stress chaperone

We cloned, expressed, and purified the hdeB gene product, which belongs to the hdeAB acid stress operon. We extracted HdeB from bacteria by the osmotic-shock procedure and purified it to homogeneity by ion-exchange chromatography and hydroxyapatite chromatography. Its identity was confirmed by mass spectrometry analysis. HdeB has a molecular mass of 10 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which matches its expected molecular mass. We purified the acid stress chaperone HdeA in parallel in order to compare the two chaperones. The hdeA and hdeB mutants both display reduced viability upon acid stress, and only the HdeA/HdeB expression plasmid can restore their viability to close to the wild-type level, suggesting that both proteins are required for optimal protection of the bacterial periplasm against acid stress. Periplasmic extracts from both mutants aggregate at acidic pH, suggesting that HdeA and HdeB are required for protein solubilization. At pH 2, the aggregation of periplasmic extracts is prevented by the addition of HdeA, as previously reported, but is only slightly reduced by HdeB. At pH 3, however, HdeB is more efficient than HdeA in preventing periplasmic-protein aggregation. The solubilization of several model substrate proteins at acidic pH supports the hypothesis that, in vitro, HdeA plays a major role in protein solubilization at pH 2 and that both proteins are involved in protein solubilization at pH 3. Like HdeA, HdeB exposes hydrophobic surfaces at acidic pH, in accordance with the appearance of its chaperone properties at acidic pH. HdeB, like HdeA, dissociates from dimers at neutral pH into monomers at acidic pHs, but its dissociation is complete at pH 3 whereas that of HdeA is complete at a more acidic pH. Thus, we can conclude that Escherichia coli possesses two acid stress chaperones that prevent periplasmic-protein aggregation at acidic pH.     

Baroreflex control of renal sympathetic nerve activity during air-jet stress in rats

The effects of acute emotional stress on the sympathetic component of the arterial baroreceptor reflex have not yet been described in conscious animals and humans. Arterial pressure (AP) and renal sympathetic nerve activity (RSNA) were simultaneously recorded in 11 conscious rats before and during exposure to a mild environmental stressor (jet of air). Baroreflex function curves relating AP and RSNA were constructed by fitting a sigmoid function to RSNA and AP measured during sequential nitroprusside and phenylephrine administrations. Stress increased mean AP from 112 +/- 2 to 124 +/- 2 mmHg, heart rate from 381 +/- 10 to 438 +/- 18 beats/min, and RSNA from 0.80 +/- 0.14 to 1.49 +/- 0.23 microV. The RSNA-AP relationship was shifted toward higher AP values, and its maximum gain was significantly (P < 0.01) increased from 9.0 +/- 1.3 to 16.2 +/- 2.1 normalized units (NU)/mmHg. The latter effect was secondary to an increase (P < 0.01) in the range of the RSNA variation from 285 +/- 33 to 619 +/- 59 NU. In addition, the operating range of the reflex was increased (P < 0.01) from 34 +/- 2 to 41 +/- 3 mmHg. The present study indicates that in rats, the baroreflex control of RSNA is sensitized and operates over a larger range during emotional stress, which suggests that renal vascular tone, and possibly AP, are very efficiently controlled by the sympathetic nervous system under this condition.     

A transfer function method for the continuous assessment of baroreflex control of renal sympathetic nerve activity in rats

The present study examined whether the gain of the transfer function relating cardiac-related rhythm of renal sympathetic nerve activity (RSNA) to arterial pressure (AP) pulse might serve as a spontaneous index of sympathetic baroreflex sensitivity (BRS). AP and RSNA were simultaneously recorded in conscious rats, either baroreceptor-intact (control, n = 11) or with partial denervation of baroreflex afferents [aortic baroreceptor denervated (ABD; n = 10)] during 1-h periods of spontaneous activity. Transfer gain was calculated over 58 adjacent 61.4-s periods (segmented into 10.2-s periods). Coherence between AP and RSNA was statistically (P < 0.05) significant in 90 +/- 3% and 56 +/- 10% of cases in control and ABD rats, respectively. Transfer gain was higher (P = 0.0049) in control [2.39 +/- 0.13 normalized units (NU)/mmHg] than in ABD (1.48 +/- 0.22 NU/mmHg) rats. In the pooled study sample, transfer gain correlated with sympathetic BRS estimated by the vasoactive drug injection technique (R = 0.75; P < 0.0001) and was inversely related to both time- (standard deviation; R = -0.74; P = 0.0001) and frequency-domain [total spectral power (0.00028-2.5 Hz); R = -0.82; P < 0.0001] indices of AP variability. In control rats, transfer gain exhibited large fluctuations (coefficient of variation: 34 +/- 3%) that were not consistently related to changes in the mean level of AP, heart rate, or RSNA. In conclusion, the transfer function method provides a continuous, functionally relevant index of sympathetic BRS and reveals that the latter fluctuates widely over time.     

Osteoblast Menin Regulates Bone Mass in Vivo

Menin, the product of the multiple endocrine neoplasia type 1 (Men1) tumor suppressor gene, mediates the cell proliferation and differentiation actions of transforming growth factor-β (TGF-β) ligand family members. In vitro, menin modulates osteoblastogenesis and osteoblast differentiation promoted and sustained by bone morphogenetic protein-2 (BMP-2) and TGF-β, respectively. To examine the in vivo function of menin in bone, we conditionally inactivated Men1 in mature osteoblasts by crossing osteocalcin (OC)-Cre mice with floxed Men1 (Men1^f/f) mice to generate mice lacking menin in differentiating osteoblasts (OC-Cre;Men1^f/f mice). These mice displayed significant reduction in bone mineral density, trabecular bone volume, and cortical bone thickness compared with control littermates. Osteoblast and osteoclast number as well as mineral apposition rate were significantly reduced, whereas osteocyte number was increased. Primary calvarial osteoblasts proliferated more quickly but had deficient mineral apposition and alkaline phosphatase activity. Although the mRNA expression of osteoblast marker and cyclin-dependent kinase inhibitor genes were all reduced, that of cyclin-dependent kinase, osteocyte marker, and pro-apoptotic genes were increased in isolated Men1 knock-out osteoblasts compared with controls. In contrast to the knock-out mice, transgenic mice overexpressing a human menin cDNA in osteoblasts driven by the 2.3-kb Col1a1 promoter, showed a gain of bone mass relative to control littermates. Osteoblast number and mineral apposition rate were significantly increased in the Col1a1-Menin-Tg mice. Therefore, osteoblast menin plays a key role in bone development, remodeling, and maintenance.     

Peptidase activity of the Escherichia coli Hsp31 chaperone

Hsp31, the Escherichia coli hcha gene product, is a molecular chaperone whose activity is inhibited by ATP at high temperature. Its crystal structure reveals a putative Cys(184), His(185), and Asp(213) catalytic triad similar to that of the Pyrococcus horikoshii protease PH1704, suggesting that it should display a proteolytic activity. A preliminary report has shown that Hsp31 has an exceedingly weak proteolytic activity toward bovine serum albumin and a peptidase activity toward two peptide substrates with small amino acids at their N terminus (alanine or glycine), but the physiological significance of this observation remains unclear. In this study, we report that Hsp31 does not diplay any significant proteolytic activity but has peptidolytic activity. The aminopeptidase cleavage preference of Hsp31 is Ala > Lys > Arg > His, suggesting that Hsp31 is an aminopeptidase of broad specificity. Its aminopeptidase activity is inhibited by the thiol reagent iodoacetamide and is completely abolished in a C185A mutant, which is consistent with Hsp31 being a cysteine peptidase. The aminopeptidase activity of Hsp31 is also inhibited by EDTA and 1,10-phenanthroline, in concordance with the importance of the putative His(85), His(122), and Glu(90) metal-binding site revealed by crystallographic studies. An Hsp31-deficient mutant accumulates more 8-12-mer peptides than its parental strain, and purified Hsp31 can transform these peptides into smaller peptides, suggesting that Hsp31 has an important peptidase function both in vivo and in vitro. Proteins interacting with Hsp31 have been identified by reverse purification of a crude E. coli extract on an Hsp31-affinity column, followed by SDS-polyacrylamide electrophoresis and mass spectrometry. The ClpA component of the ClpAP protease, the chaperone GroEL, elongation factor EF-Tu, and tryptophanase were all found to interact with Hsp31, thus substantiating the role of Hsp31 as both chaperone and peptidase.     

Baroreflex control of lumbar and renal sympathetic nerve activity in conscious rats

This study compared the baroreflex control of lumbar and renal sympathetic nerve activity (SNA) in conscious rats. Arterial pressure (AP) and lumbar and renal SNA were simultaneously recorded in six freely behaving rats. Pharmacological estimates of lumbar and renal sympathetic baroreflex sensitivity (BRS) were obtained by means of the sequential intravenous administration of sodium nitroprusside and phenylephrine. Sympathetic BRS was significantly (P < 0.05) lower for lumbar [3.0 +/- 0.4 normalized units (NU)/mmHg] than for renal (7.6 +/- 0.6 NU/mmHg) SNA. During a 219-min baseline period, spontaneous lumbar and renal BRS were continuously assessed by computing the gain of the transfer function relating AP and SNA at heart rate frequency over consecutive 61.4-s periods. The transfer gain was considered only when coherence between AP and SNA significantly differed from zero, which was verified in 99 +/- 1 and 96 +/- 3% of cases for lumbar and renal SNA, respectively. When averaged over the entire baseline period, spontaneous BRS was significantly (P < 0.05) lower for lumbar (1.3 +/- 0.2 NU/mmHg) than for renal (2.3 +/- 0.3 NU/mmHg) SNA. For both SNAs, spontaneous BRS showed marked fluctuations (variation coefficients were 26 +/- 2 and 28 +/- 2% for lumbar and renal SNA, respectively). These fluctuations were positively correlated in five of six rats (R = 0.44 +/- 0.06; n = 204 +/- 8; P < 0.0001). We conclude that in conscious rats, the baroreflex control of lumbar and renal SNA shows quantitative differences but is modulated in a mostly coordinated way.     

Ectomycorrhizal communities of Quercus garryana are similar on serpentine and nonserpentine soils

Serpentine soils, rich in iron, magnesium, and heavy metals, select for unique plant communities and for endemic species. Because mycorrhizal fungi mediate the interaction between plants and soil, we hypothesized that distinct ectomycorrhizal fungi would colonize Quercus garryana roots on serpentine and nonserpentine soils. We sampled roots of Q. garryana on serpentine soils at two locations in the Klamath-Siskiyou Mountains of southwestern Oregon and identified ectomycorrhizas by morphological and molecular methods. The same six most abundant and most frequent mycorrhizal species, Cenococcum geophilum, Tuber candidum, Genea harknessii, Tomentella sp., Sebacina sp., and Inocybe sp., were found on serpentine and nonserpentine soils. Based on similarities calculated using the Sørensen index in Non-metric Multidimensional Scaling, mycorrhizal communities on serpentine and nonserpentine soils were not significantly different. This study showed that ectomycorrhizal species associated with Q. garryana exhibit edaphic tolerance and were neither reduced nor excluded by serpentinite or peridotite parent materials.