Joseph P. Stains, PhD

The research in my lab examines signaling and gene transcription in cells of the skeletal system, and how signaling networks affect osteogenic differentiation, bone formation and bone quality.

GAP JUNCTIONS IN BONE

A major project in my lab explores the molecular details of how osteogenic cells use cell-to-cell signaling through connexin43-containing gap junctions to coordinate function using in vitro and in vivo models. 

Gap junctions are intercellular channels formed by hexamers of connexins in one cell that dock with a hexameric array of connexins on an adjacent cell, forming an aqueous pore between the two cells. Gap junctions permit the direct intercellular exchange of small molecules, such as ions, metabolites and second messengers.

In bone, osteoblasts and osteocytes are highly interconnected via gap junctions composed primarily of connexin43. In these cells, connexin43 has been shown to play an important role in transmitting hormonal-, mechanical load- and growth factor-induced signals and, ultimately, in bone mass acquisition. In addition, mutations in Gja1, the gene encoding Cx43, result in the pleiotropic disorder oculodentodigital dysplasia, which includes skeletal manifestations.

Despite the clear importance of connexin43 in skeletal function, key molecular details of how connexin43 regulates bone mass acquisition, osteoblast differentiation and osteoblast/osteocyte function are unknown. My work is focused on answering key questions, such as what are the identities of the second messengers communicated by connexin43 gap junctions, what are the targets of these second messengers, and how do they regulate osteoblast/osteocyte function?

In my lab, we use in vivo models, as well as molecular, biochemical and cell biological approaches to determine the role of connexin43 in bone and to elucidate the molecular mechanisms underpinning the impact of connexin43 on osteoblast gene transcription, signal transduction and bone formation.

OSTEOCYTE MECHANOTRASNDUCTION AND THE CYTOSKELETON

A recent focus of my lab has been on the role of cytoskeletal proteins in the mechanically induced response observed in bone cells following mechanical loading. Bone dynamically remodels to adapt to mechanical loads to maintain its structural integrity. Bone-embedded osteocytes that reside in the fluid-filled lacunar-canalicular system are central to skeletal mechanoresponsiveness. We have been intersted in how these cells sense and respond to these mechanical cues to orchestrate bone remodeling and repair.

We have recently mechanistically linked fluid shear stress induced Ca²⁺influx to the degradation of sclerostin, a key regulator of bone formation. We have defined a specific subset of post-translationally modified microtubules that serve as rheostats, controlling the stiffness of the cytoskeleton and tuning the cells response to mechanical cues. Specifically, we have defined a mechanically activated NOX2-ROS-TRPV4-Ca²⁺-CaMKII pathway that leads to the rapid degradation of sclerostin protein.

During this work, we developed and patented a novel device to interrogate cell responses to fluid shear stress by both cell imaging and molecular techniques in the same device. Additionally, we collaborated on an investigated the role of the cytoskeletal regulator MARK3 on bone and examined the contribution of the multifunctional intermediate filament protein, synemin, to bone mass. 

Gap Junctions

(A) Gap junctions are composed of connexin monomers. Six connexin monomers assemble to form a connexon or hemichannel in the plasma membrane of one cell. A hemichannel can then dock with a hemichannel on an adjacent cell to form a permeable channel between the cells. (B) Immunofluorescence of the gap junction protein connexin43 in osteoblast cells. Connexin43, green; actin, red; nuclei, blue. (C) Gap junctional communication (the exchange of low molecular weight molecules) between osteoblasts can be visualized by the scrape loading dye transfer assay. (D) Immunohistochemistry of connexin43 (reddish-brown) in osteoblasts during bone formation.

Current Research Support:

03/01/2013-2/28/2025 "Spatial Control of Bone Remodeling by Gap Junction-Communicated cAMP" NIH/NIAMS R01 AR063631. Role: PI

3/01/2018-2/28/2023  “Mechanisms of osteocyte mechano-signaling and sclerostin regulationl” NIH NIAMS R01 AR071614. Role: PI (MPI)

Completed Research Support:

4/01/2019-3/31/2020 "Microtubules and Mechanobiology in the Crosstalk of Bone and White Adipose Tissue" Mid-Atlantic Nutrition Obesity Research Center Pilot & Feasibility Grant

10/01/2016-9/30/2017 “h-MSC Based Delivery of siRNAs to Chondrocytes and Synovial Tissue in a Rat Model” Pilot and Feasibility (P&F) Grant from the Baltimore Center for Musculoskeletal Research. Role Co-I

10/01/2015-9/30/2016 “Microtubule Dependent Mechanotransduction in Bone” Pilot and Feasibility (P&F) Grant from the Baltimore Center for Musculoskeletal Research. Role Co-I

07/01/2013-06/30/2016 "Optimal Treatment of Malignant Long Bone Fracture: Influence of Method of Repair and External Beam Irradiation on the Pathway and Efficacy of Fracture Healing" DoD PR120168. Role: Co-I

07/01/2014-06/30/2016 "The Role of the beta-Catenin Signaling Cascade in the Skeletal Phenotype of Hutchinson Gilford Progeria Syndrome" TEDCO/Maryland Stem Cell Research Fund, 2014-MSCRFE-0645. Role; PI

7/01/2012-6/30/2014 "Using Gap Junctions to Enhance Stem Cell Therapies in Osteoarthritis" TEDCO/MSCRF 2012-MSCRFE-0154-00. Role: PI

5/01/2006-8/30/2011 "Intercellular Signaling in Bone" NIH/NIAMS R01 AR052719. Role: PI

Real time PCR, luciferase reporter assays, western blotting, cell transfections, cell culture, chromatin immunoprecipitations (ChIP), fluorescence microscopy, immunohistochemistry, skeletal histology, bone histomorphometry, whole mount staining and micro computed tomography.

(A) Immunofluorescence of cytoskeleton proteins in osteocytes. (B) Sub-micron resolution microCT slice of a mouse femoral diaphysis. (C) Whole mount Alizarin Red/Alcian blue stained neonatal mouse. (D) Immunofluorescence of gap junctions (green) and actin (red) in cultured osteoblasts. (E) MicroCT scan of a mouse skull. (F) Trichrome stain of the mouse distal femur.

Orthopaedics Research at the University of Maryland